Methods of detecting a polypeptide having anaplastic lymphoma kinase activity in kidney cancer

ABSTRACT

The invention provides methods to identify, diagnose, and treat kidney cancer through the detection of expression and/or activity of anaplastic lymphoma kinase (ALK). The detection of the presence of a polypeptide with ALK kinase activity (e.g., by detecting expression and/or activity of the polypeptide), identify those kidney cancers that are likely to respond to an ALK-inhibiting therapeutic.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/844,832, filed Sep. 3, 2015, which is a continuation of U.S. patentapplication Ser. No. 13/204,342, filed Aug. 5, 2011, now abandoned,which claims priority to U.S. Provisional Application Ser. No.61/371,525, filed Aug. 6, 2010, the entire contents of which are herebyincorporated by reference.

FIELD OF THE INVENTION

The invention relates generally to proteins and genes involved in cancer(e.g., kidney cancer), and to the detection, diagnosis and treatment ofcancer.

BACKGROUND OF THE INVENTION

Many cancers are characterized by disruptions in cellular signalingpathways that lead to aberrant control of cellular processes, or touncontrolled growth and proliferation of cells. These disruptions areoften caused by changes in the activity of particular signalingproteins, such as kinases.

Aberrant expression of protein kinase proteins can be the causativeagent of (and the driver of) cancer. Aberrant expression can be causedby the fusion of the protein (or kinase portion thereof) with asecondary protein (or portion there), expression of a truncated portionof the protein, or by abnormal regulation of expression of thefull-length protein.

It is known that gene translocations resulting in kinase fusion proteinswith aberrant signaling activity can directly lead to certain cancers(see, e.g., Mitelman et al., Nature Reviews Cancer 7: 233-245, 2007,Futreal et al., Nat Rev Cancer 4(3): 177-183 (2004), and Falini et al.,Blood 99(2): 409-426 (2002). For example, it has been shown that theBCR-ABL oncoprotein, a tyrosine kinase fusion protein, is the causativeagent and drives human chronic myeloid leukemia (CML). The BCR-ABLoncoprotein, which is found in at least 90-95% of CML cases, isgenerated by the translocation of gene sequences from the c-ABL proteintyrosine kinase on chromosome 9 into BCR sequences on chromosome 22,producing the so-called Philadelphia chromosome. See, e.g. Kurzock etal., N. Engl. J. Med. 319: 990-998 (1988). The translocation is alsoobserved in acute lymphocytic leukemia (ALL) and acute myeloid leukemia(AML) cases. These discoveries spurred FDA approval of imatinib mesylate(sold under the trademark Gleevec® by Novartis) and dasatinig (sold byBristol-Mysers Squibb under the trademark Sprycel®), small moleculeinhibitors of the ABL kinase, for the treatment of CML and ALL. Thesedrugs are examples of drugs that are designed to interfere with thesignaling pathways that drive the growth of tumor cells. The developmentof such drugs represents a significant advance over the conventionaltherapies for CML and ALL, chemotherapy and radiation, which are plaguedby well known side-effects and are often of limited effect since theyfail to specifically target the underlying causes of the malignancies.

Thus, it would be useful to identify proteins that drive cancers inorder to detect cancers at an early stage, when they are more likely torespond to therapy, and for the development of new reagents and methodsfor the study, diagnosis, and treatment of such cancers. Additionally,identification of such proteins will, among other things, desirablyenable new methods for selecting patients for targeted therapies, aswell as for the screening and development of new drugs that inhibit suchproteins and, thus, treat cancer.

SUMMARY OF THE INVENTION

The invention is based on the discovery of ALK kinase in kidney cancercells. This unexpected discovery enables methods to detect, treat, andcure kidney cancers driven by ALK kinase activity.

In a first aspect, the invention provides a method for detecting thepresence and/or activity of a polypeptide with ALK kinase activity in abiological sample from a mammalian kidney cancer or suspected mammaliankidney cancer. The method includes (a) obtaining a biological samplefrom a mammalian kidney cancer or suspected mammalian kidney cancer and(b) contacting the biological sample with a detection molecule selectedfrom the group consisting of a reagent that detects ALK kinase activity,a reagent that detects a polypeptide with ALK kinase activity, and areagent that detects to a polynucleotide encoding the polypeptide withALK kinase activity, and (c) detecting reaction of the detectionmolecule with the biological sample, wherein reaction of the detectionmolecule with the biological sample indicates said polypeptide with ALKkinase activity is present or active in said mammalian kidney cancer orsuspected mammalian kidney cancer. In some embodiments, the detectionmolecule is detectably labeled.

In another aspect, the invention provides a method for identifying amammalian kidney cancer or suspected mammalian kidney cancer thatbelongs to a subset of kidney cancers driven by ALK kinase activity,said method comprising the steps of (a) contacting a biological sampleobtained from a mammalian kidney cancer or suspected mammalian kidneycancer with at least one detection molecule selected from the groupconsisting of a reagent that detects ALK kinase activity, a reagent thatdetects a polypeptide with ALK kinase activity, and a reagent thatdetects to a polynucleotide encoding the polypeptide with ALK kinaseactivity, and (b) detecting reaction of the detection molecule with saidbiological sample, wherein the reaction of the detection molecule withsaid biological sample indicates that said mammalian kidney cancer orsuspected mammalian kidney cancer is driven by ALK kinase activity. Insome embodiments, the mammalian kidney cancer or suspected mammaliankidney cancer driven by ALK kinase activity is likely to respond to acomposition comprising at least one ALK-inhibiting therapeutic.

In another aspect, the invention provides method for determining whethera compound inhibits the progression of a mammalian kidney cancer orsuspected mammalian kidney cancer driven by a polypeptide with ALKkinase activity, said method comprising the step of determining whethersaid compound inhibits the expression and/or activity of saidpolypeptide in said cancer mammalian kidney cancer or suspectedmammalian kidney cancer.

In another aspect, the invention provides a method for inhibiting theprogression of a mammalian kidney cancer or suspected mammalian kidneycancer driven by polypeptide with ALK kinase activity, comprisinginhibiting the expression and/or activity of said polypeptide in saidmammalian kidney cancer or suspected mammalian kidney cancer.

In another aspect, the invention provides a method for treating amammalian patient with mammalian kidney cancer or suspected mammaliankidney cancer driven by a polypeptide with ALK kinase activity, saidmethod comprising the step of administering a composition comprising atherapeutically effective amount of a composition comprising anALK-inhibiting therapeutic to the mammalian patient.

In still a further aspect, the invention provides a method for treatinga patient having a mammalian kidney cancer or suspected mammalian kidneycancer comprising the steps of: (a) detecting the presence or activityof said polypeptide with ALK kinase activity a biological sample of themammalian kidney cancer or suspected mammalian kidney cancer of thepatient; and (b) administering a composition comprising anALK-inhibiting therapeutic to the patient. In some embodiments, thepatient is a human.

In various embodiments of all of the aspects of the invention, themethod for detecting the presence and/or activity of a polypeptide withALK kinase activity in a biological sample from a mammalian kidneycancer or suspected mammalian kidney cancer comprising the steps of: (a)obtaining a biological sample from a mammalian kidney cancer orsuspected mammalian kidney cancer and (b) contacting the biologicalsample with a reagent that detects ALK kinase activity, whereindetection of ALK kinase activity by the reagent in the biological sampleindicates said polypeptide with ALK kinase activity is present in saidbiological sample. In some embodiments, the method for detecting thepresence and/or activity of a polypeptide with ALK kinase activity in abiological sample from a mammalian kidney cancer or suspected mammaliankidney cancer comprises the steps of: (a) obtaining a biological samplefrom a mammalian kidney cancer or suspected mammalian kidney cancer and(b) contacting the biological sample with a reagent that detects apolypeptide with ALK kinase activity, wherein detection of saidpolypeptide in said biological sample indicates said polypeptide withALK kinase activity is present in said biological sample. In someembodiments, the method for detecting the presence and/or activity of apolypeptide with ALK kinase activity in a biological sample from amammalian kidney cancer or suspected mammalian kidney cancer comprisesthe steps of: (a) obtaining a biological sample from a mammalian kidneycancer or suspected mammalian kidney cancer and (b) contacting thebiological sample with a reagent that detects a polynucleotide encodingthe polypeptide with ALK kinase activity, wherein detection of saidpolynucleotide in said biological sample indicates said polypeptide withALK kinase activity is present in said biological sample.

In various embodiments of all of the aspects of the invention, theinhibition of the expression and/or activity of the polypeptide isdetermined using a reagent that detects ALK kinase activity, a reagentthat detects a polypeptide with ALK kinase activity, or a reagent thatdetects to a polynucleotide encoding the polypeptide with ALK kinaseactivity.

In various embodiments of all of the aspects of the invention, thedetection molecule is detectably labeled.

In various embodiments of all of the aspects of the invention, themammalian kidney cancer or suspected mammalian kidney cancer in whichthe presence or activity of said polypeptide with ALK kinase activity isdetected is identified as a mammalian kidney cancer or suspectedmammalian kidney cancer belonging to a subset of kidney cancers drivenby ALK kinase activity. In some embodiments, the mammalian kidney canceror suspected mammalian kidney cancer in which the presence or activityof said polypeptide with ALK kinase activity is detected is identifiedas a mammalian kidney cancer or suspected mammalian kidney cancer likelyto respond to an ALK-inhibiting therapeutic.

In various embodiments of all of the aspects of the invention, thepolypeptide with ALK kinase activity is aberrantly expressed full-lengthALK protein. In various embodiments, the polypeptide with ALK kinaseactivity is an ALK fusion polypeptide comprising at least a portion of afirst fusion member and at least a portion of a second fusion member,wherein the second fusion member is an ALK protein comprises an ALKkinase domain. In some embodiments, the portion of the ALK protein. Insome embodiments, the first fusion member is an NPM polypeptide, a ALO17polypeptide, a TFG polypeptide, a MSN polypeptide, a TPM3 polypeptide, aTPM4 polypeptide, an ATIC polypeptide, a MYH9 polypeptide, a CLTCpolypeptide, a SEC31L1 polypeptide, an RANBP2 polypeptide, a CARSpolypeptide, an EML4 polypeptide, a KIF5B polypeptide, or a VCLpolypeptide. In some embodiments, the ALK fusion polypeptide is anNPM-ALK fusion polypeptide, an ALO17-ALK fusion polypeptide, an MSN-ALKfusion polypeptide, an TPM3-ALK fusion polypeptide, an TPM4-ALK fusionpolypeptide, an ATIC-ALK fusion polypeptide, an MYH9-ALK fusionpolypeptide, an CLTC-ALK fusion polypeptide, an SEC31L1-ALK fusionpolypeptide, an RANBP2-ALK fusion polypeptide, an CARS-ALK fusionpolypeptide, an EML4-ALK fusion polypeptide, an KIF5B-ALK fusionpolypeptide, a TFG-ALK fusion polypeptide, or a VCL-ALK fusionpolypeptide.

In various embodiments of all of the aspects of the invention, thepolypeptide with ALK kinase activity is a truncated ALK polypeptide. Insome embodiments, the reagent that detects ALK kinase activity is asubstrate of ALK. In some embodiments, the method is implemented in anin vitro kinase assay format. In some embodiments, the method isimplemented in an immunological assay employing a phosphorylatedtyrosine-specific binding agent (e.g., an antibody that specificallybinds to phosphorylated tyrosine residues).

In various embodiments of all of the aspects of the invention, thereagent that detects a polypeptide with ALK kinase activity is a reagentthat specifically binds to the polypeptide. In some embodiments, thereagent that specifically binds to the polypeptide is an antibody. Insome embodiments, the reagent that specifically binds to the polypeptideis an AQUA peptide. In some embodiments, the antibody specifically bindsto a full length ALK protein.

In various embodiments of all of the aspects of the invention, where theprotein with ALK kinase activity is a ALK fusion polypeptide, thereagent that detects a polypeptide with ALK kinase activity is reagentthat specifically binds the ALK fusion polypeptide. In some embodiments,the reagent that specifically binds to the ALK fusion polypeptide is anantibody. In some embodiments, the reagent that specifically binds tothe ALK fusion polypeptide is an AQUA peptide. In some embodiments, theantibody specifically binds to the portion of the ALK protein present inthe ALK fusion, to the portion of the first fusion member present in theALK fusion, or to a junction between the first fusion member and theportion of the ALK protein present in the ALK fusion.

In various embodiments of all of the aspects of the invention, themethod is implemented in a flow cytometry assay format, animmunohistochemistry (IHC) assay format, an immunofluorescence (IF)assay format, an Enzyme-linked immunosorbent assay (ELISA) assay format,a Western blotting analysis assay format, or a mass spectrometry assayformat.

In various embodiments of all of the aspects of the invention, thereagent that detects a polynucleotide encoding the polypeptide with ALKkinase activity is a nucleic acid molecule that hybridizes to saidpolynucleotide. In some embodiments, the nucleic acid moleculehybridizes to the polynucleotide under stringent conditions.

In various embodiments of all of the aspects of the invention, thenucleic acid molecule is a polymerase chain reaction (PCR) probe, afluorescence in situ hybridization (FISH) probe, or a Southern blottingprobe. In some embodiments, the method is implemented in a polymerasechain reaction (PCR) assay format, a in situ hybridization (ISH) assayformat, or a Southern blotting assay format.

In various embodiments of all of the aspects of the invention, themammalian kidney cancer or suspected mammalian kidney cancer is agranular cell cancer or a squamous cell cancer. In various embodiments,the mammalian kidney cancer or suspected mammalian kidney cancer is froma mammal. In various embodiments, the mammalian kidney cancer orsuspected mammalian kidney cancer is from a human.

In various embodiments of all of the aspects of the invention, thebiological sample is a circulating tumor cell from said mammalian kidneycancer or suspected mammalian kidney cancer. In certain embodiments, thebiological sample is a tissue biopsy or a fine needle aspirate from saidmammalian kidney cancer or suspected mammalian kidney cancer.

In various embodiments of all of the aspects of the invention, a patientfrom whom said biological sample is obtained is diagnosed as having amammalian kidney cancer or suspected mammalian kidney cancer driven bythe polypeptide with ALK kinase activity. In some embodiments, thepatient is administered pharmaceutical composition comprising anALK-inhibiting therapeutic (e.g., PF-02341066, NVT TAE-684, AP26113,CEP-14083, CEP-14513, CEP11988, WHI-P131 or WHI-P154).

In various embodiments of all of the aspects of the invention, theexpression and/or activity of the polypeptide with ALK kinase activityis inhibited with a composition comprising an ALK-inhibitingtherapeutic. In some embodiments, the ALK-inhibiting therapeutic isPF-02341066 (also known as crizotinib). In some embodiments, theALK-inhibiting therapeutic is NVT TAE-684, AP26113, or CEP-14083,CEP-14513, CEP11988, WHI-P131 and WHI-P154.

In still another aspect, the invention provides methods for diagnosingkidney cancer in a patient (also referred to as a subject). The methodscomprise obtaining a biological sample from a subject suspected ofhaving kidney cancer, obtaining a control biological sample from anormal individual not suspected of having kidney cancer, measuring thelevel of expression and/or activity of a polypeptide with ALK kinaseactivity or polynucleotide encoding a polypeptide with ALK kinaseactivity in the biological sample and control biological sample using adetection device, generating a database of the detected levels ofexpression and/or activity of said polypeptide with ALK kinase activityor polynucleotide in the biological sample and control biologicalsample, and obtaining a report from the database of the detection devicewherein a higher level of expression and/or activity of the polypeptidewith ALK kinase activity or polynucleotide in the biological samplerelative to the control biological sample is correlated to a kidneycancer diagnosis. Suitably, the kidney cancer is likely to respond totreatment with an ALK inhibitor (i.e., an ALK-inhibiting therapeutic).

In certain embodiments, the polynucleotide encoding a polypeptide withALK kinase activity is an mRNA or cDNA encoding full-length ALK proteinor encoding an ALK fusion polypeptide, including a ALK fusionpolypeptide comprising the intracellular domain of ALK, the tyrosinekinase domain of ALK, or the C-terminal domain of ALK. Detecting of themRNA can be performed by any method including, for example, by RT-PCR orNorthern blot analysis.

The polypeptide with ALK kinase activity can be detected by an antibodyspecific for the intracellular domain of ALK, including an antibody thatdoes not cross-react with c-Met, and an antibody that is specific forthe tyrosine kinase domain of ALK.

In another embodiment, the polypeptide with ALK kinase activity can bedetected using a reagent that is specific for an ALK fusion polypeptidecomprising amino acids 1376-1620 of full length ALK (where full lengthALK is 1620 amino acids and is set forth in SEQ ID NO: 2), amino acidresidues 1504-1507 of full length ALK, or amino acid residues 1603-1606of full length ALK.

In embodiments, the ALK fusion polypeptide is selected from the groupconsisting of VCL-ALK, EML4-ALK, NPM-ALK, TPM3-ALK, TFG-ALK, ATIC-ALK,CLTC-ALK, MSN-ALK, TPM4-ALK, ALO17-ALK, RANBP2-ALK, MYH9-ALK, CARS-ALK,SEC31L1-ALK, and KIF5B-ALK.

In additional aspects, the invention provides methods for treating akidney cancer in a patient (also referred to as a subject). The methodscomprise obtaining a biological sample from a patient suspected ofhaving kidney cancer, obtaining a control biological sample from anormal individual not suspected of having kidney cancer, detecting alevel of expression and/or activity of a polypeptide with ALK kinaseactivity in the biological sample and the control biological sample andtreating the subject with an ALK inhibitor if the level of expressionand/or activity of the polypeptide with ALK kinase activity in thebiological sample is greater than the level of expression and/oractivity of the polypeptide with ALK kinase activity in the controlbiological sample.

In further aspects, the invention provides methods for diagnosing kidneycancer in a subject comprising obtaining a biological sample from apatient suspected of having kidney cancer and determining whether apolypeptide with ALK kinase activity or polynucleotide encoding the sameis present in the biological sample.

The invention also provides methods for treating a kidney cancer in apatient comprising obtaining a biological sample from a patientsuspected of having kidney cancer, determining whether a polypeptidewith ALK kinase activity or polynucleotide encoding the same is presentin the biological sample and treating the patient with an ALK inhibitorif the polypeptide with ALK kinase activity or polynucleotide encodingthe same is present.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is an immunohistochemical representation demonstratingALK-specific monoclonal antibody staining of squamous cell carcinoma ofthe kidney.

FIG. 2 is an immunohistochemical representation demonstratingALK-specific monoclonal antibody staining of granular cell carcinoma ofthe kidney.

FIG. 3 is an image (with the inset showing a expanded view of the cellimmediately to the upper right of the inset) depicting the detection ofALK by FISH assay employing a dual-color (orange/green) break-apartprobe specific to the ALK locus in kidney squamous cells. The yellowarrows point to orange/green signals either immediately adjacent to eachother or fused together confirming the presence of the 2p23 ALK regionin its native state. The red arrows point to a rearranged ALK locus.Images were taken at 100× magnification with digital zoom inset.

FIG. 4A depicts a Western blot of MDCK cell lysates probed withantibodies directed to pMet Y1234/5 antibody (Cell Signaling Technology,Inc., Danvers, Mass.; Catalog #3077), Met (25H2) mouse monoclonalantibody (Cell Signaling Technology #3127), pALK Y1278/82/83 antibody(Cell Signaling Technology, #3983), ALK (D5F3) XP®RmAb (Cell SignalingTechnology #3633), and Beta Actin (Cell Signaling Technology #4970)after a 1 second exposure. The lanes represented MDCK serum starvedovernight (lane 1), MDCK serum starved overnight, followed by HGFstimulation (50 ng/ml) for 5 minutes (lane 2), MDCK serum starvedovernight, then serum starved over 24 hours (lane 3), MDCK serum starvedfollowed by HGF stimulation (50 ng/ml) for 24 hours (lane 4), H3122cells (positive control for ALK (EML4-ALK v1))(lane 5), and MKN45 cells(positive control for pMet)(lane 6) are depicted.

FIG. 4B depicts a Western blot of MDCK cell lysates probed withantibodies directed to pMet Y1234/5 antibody (Cell Signaling Technology,#3077), Met (25H2) mouse monoclonal antibody (Cell Signaling Technology,#3127), pALK Y1278/82/83 antibody (Cell Signaling Technology #3983), ALK(D5F3) XP®RmAb (Cell Signaling Technology #3633), and Beta Actin (CellSignaling Technology #4970) after a 15 second exposure. The lanesrepresented MDCK serum starved overnight (lane 1), MDCK serum starvedovernight, followed by HGF stimulation (50 ng/ml) for 5 minutes (lane2), MDCK serum starved overnight, then serum starved over 24 hours (lane3), MDCK serum starved followed by HGF stimulation (50 ng/ml) for 24hours (lane 4), H3122 cells (positive control for ALK (EML4-ALKv1))(lane 5), and MKN45 cells (positive control for pMet)(lane 6) aredepicted.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention is based upon the unexpected discovery of ALK kinase inhuman kidney cancer. As ALK kinase is not known to be expressed innormal kidney tissue and cells, the presence of ALK kinase (and ALKkinase activity) is expected to drive the proliferation and survival ofthe subset of kidney cancer in which it is expressed. Such kidneycancers may be identified (e.g., diagnosed) and/or treated in accordancewith the teachings provided herein.

Based on these discoveries, a patient whose kidney cancer (or suspectedkidney cancer) expresses a polypeptide with ALK activity where healthypatients (i.e., non-cancerous patients) do not express such proteinswith ALK activity in their normal kidney tissue may respond favorably toadministration of an ALK inhibitor (e.g., the growth of the kidneycancer may slow or stop as compared to an untreated patient sufferingfrom the same cancer).

The published patents, patent applications, websites, company names, andscientific literature referred to herein establish the knowledge that isavailable to those with skill in the art and are hereby incorporated byreference in their entirety to the same extent as if each wasspecifically and individually indicated to be incorporated by reference.Any conflict between any reference cited herein and the specificteachings of this specification shall be resolved in favor of thelatter.

The further aspects, advantages, and embodiments of the invention aredescribed in more detail below. The patents, published applications, andscientific literature referred to herein establish the knowledge ofthose with skill in the art and are hereby incorporated by reference intheir entirety to the same extent as if each was specifically andindividually indicated to be incorporated by reference. Any conflictbetween any reference cited herein and the specific teachings of thisspecification shall be resolved in favor of the latter. Likewise, anyconflict between an art-understood definition of a word or phrase and adefinition of the word or phrase as specifically taught in thisspecification shall be resolved in favor of the latter. As used herein,the following terms have the meanings indicated. As used in thisspecification, the singular forms “a,” “an” and “the” specifically alsoencompass the plural forms of the terms to which they refer, unless thecontent clearly dictates otherwise. The term “about” is used herein tomean approximately, in the region of, roughly, or around. When the term“about” is used in conjunction with a numerical range, it modifies thatrange by extending the boundaries above and below the numerical valuesset forth. In general, the term “about” is used herein to modify anumerical value above and below the stated value by a variance of 20%.

Technical and scientific terms used herein have the meaning commonlyunderstood by one of skill in the art to which the present inventionpertains, unless otherwise defined. Reference is made herein to variousmethodologies and materials known to those of skill in the art. Standardreference works setting forth the general principles of recombinant DNAtechnology include Ausubel et al. CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley & Sons, New York, N.Y. (1989 and updates throughAugust 2010); Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor Laboratory Press, New York (1989); Kaufmanet al., Eds., Handbook of Molecular and Cellular Methods in Biology inMedicine, CRC Press, Boca Raton (1995); McPherson, Ed., DirectedMutagenesis: A Practical Approach, IRL Press, Oxford (1991). Standardreference works setting forth the general principles of pharmacologyinclude Goodman and Gilman's The Pharmacological Basis of Therapeutics,11th Ed., McGraw Hill Companies Inc., New York (2006); and Remington:The Science and Practice of Pharmacy, 21 Edition, Lippincott Williams &Wilkins, 2005.

In a first aspect, the invention provides a method for detecting thepresence and/or activity of a polypeptide with ALK kinase activity in abiological sample from a mammalian kidney cancer or suspected mammaliankidney cancer. The method includes (a) obtaining a biological samplefrom a mammalian kidney cancer or suspected mammalian kidney cancer and(b) contacting the biological sample with a detection molecule selectedfrom the group consisting of a reagent that detects ALK kinase activity,a reagent that detects a polypeptide with ALK kinase activity, and areagent that detects to a polynucleotide encoding the polypeptide withALK kinase activity, and (c) detecting reaction of the detectionmolecule with the biological sample, wherein reaction of the detectionmolecule with the biological sample indicates said polypeptide with ALKkinase activity is present or active in said mammalian kidney cancer orsuspected mammalian kidney cancer.

In another aspect, the invention provides a method for identifying amammalian kidney cancer or suspected mammalian kidney cancer thatbelongs to a subset of kidney cancers driven by ALK kinase activity,said method comprising the steps of (a) contacting a biological sampleobtained from a mammalian kidney cancer or suspected mammalian kidneycancer with at least one detection molecule selected from the groupconsisting of a reagent that detects ALK kinase activity, a reagent thatdetects a polypeptide with ALK kinase activity, and a reagent thatdetects to a polynucleotide encoding the polypeptide with ALK kinaseactivity, and (b) detecting reaction of the detection molecule with saidbiological sample, wherein the reaction of the detection molecule withsaid biological sample indicates that said mammalian kidney cancer orsuspected mammalian kidney cancer is driven by ALK kinase activity. Insome embodiments, the mammalian kidney cancer or suspected mammaliankidney cancer driven by ALK kinase activity is likely to respond to acomposition comprising at least one ALK-inhibiting therapeutic.

In another aspect, the invention provides method for determining whethera compound inhibits the progression of a mammalian kidney cancer orsuspected mammalian kidney cancer driven by a polypeptide with ALKkinase activity, said method comprising the step of determining whethersaid compound inhibits the expression and/or activity of saidpolypeptide in said cancer mammalian kidney cancer or suspectedmammalian kidney cancer.

In another aspect, the invention provides a method for inhibiting theprogression of a mammalian kidney cancer or suspected mammalian kidneycancer driven by polypeptide with ALK kinase activity, comprisinginhibiting the expression and/or activity of said polypeptide in saidmammalian kidney cancer or suspected mammalian kidney cancer.

In another aspect, the invention provides a method for treating amammalian patient with mammalian kidney cancer or suspected mammaliankidney cancer driven by a polypeptide with ALK kinase activity, saidmethod comprising the step of administering a composition comprising atherapeutically effective amount of a composition comprising anALK-inhibiting therapeutic to the mammalian patient.

In still a further aspect, the invention provides a method for treatinga patient having a mammalian kidney cancer or suspected mammalian kidneycancer comprising the steps of: (a) detecting the presence or activityof said polypeptide with ALK kinase activity a biological sample of themammalian kidney cancer or suspected mammalian kidney cancer of thepatient; and (b) administering a composition comprising anALK-inhibiting therapeutic to the patient. In some embodiments, thepatient is a human.

As used herein, by “reaction” as in the reaction of the detectionmolecule with a biological sample is meant that the detection moleculeis detecting its target in the biological sample. The nature of thereaction will, of course, depend upon the type of detection moleculeused. The methods of the invention include the use of detectionmolecules that may be reagents that detect ALK kinase activity (e.g., aphosphotyrosine-specific antibody that detects phosphorylation of an ALKsubstrate or detecting autophosphorylation of ALK). In this case,detection of reaction may be made by detecting specific binding of thephosphotyrosine-specific antibody to the biological sample. The methodsof the invention also include the use of detection molecules that may bereagents that detect a polypeptide with ALK kinase activity (e.g., anantibody that specifically binds the polypeptide). In this case,detection of reaction may be made by detecting specific binding of thepolypeptide with ALK kinase activity-specific antibody to the biologicalsample. The methods of the invention also include the use of detectionmolecules that may be reagents that detect a polynucleotide that encodesa polypeptide with ALK kinase activity (e.g., a nucleic acid probe thathybridizes to an exonic or intronic sequence from a portion of the ALKgene that encodes the ALK kinase domain). In this case, detection ofreaction may be made by detecting hybridization (e.g., under stringentconditions) of the probe to the biological sample.

The various aspects and embodiments of the invention are based on thediscovery of ALK in cancerous cells in the kidney.

The term “ALK” refers to Anaplastic Lymphoma Kinase. ALK (AnaplasticLymphoma Kinase) (GenBank accession Number: AB209477, UniProt AccessionNo. Q9UM73) is a receptor tyrosine kinase. This protein (which is 1620amino acids long in humans) has a transmembrane domain in the centralpart and has a carboxyl-terminal tyrosine kinase region and anamino-terminal extracellular domain (Oncogene. 1997 Jan. 30; 14 (4):439-49). See Pulford et al., Journal of Cellular Physiology,199:330-358, 2004 for a comprehensive review relating to ALK. Thefull-length ALK sequence is disclosed in U.S. Pat. No. 5,770,421. Innormal humans, full-length ALK protein expression has been detected inthe brain and central nervous system, and has been reported in the smallintestine and testis (see, e.g., Morris et al., Oncogene 14:2175-2188,1997). The amino acid sequence of full length human ALK cDNA and proteinis provided herein as SEQ ID NOs: 1 and 2, respectively. As shown inTable 1, the signal peptide, extracellular, transmembrane, and kinasedomains of ALK are found at the following amino acid residues in SEQ IDNO: 2:

TABLE 1 Domain Amino acid residues in SEQ ID NO: 2 Signal peptide  1-18Extracellular domain  19-1038 Transmembrane domain 1039-1059 Cytoplasmicdomain 1060-1620 Kinase domain 1116-1392

The polypeptide sequence of exon 20 onward of the ALK is included hereinas SEQ ID NO: 15. The polypeptide sequence of the ALK protein startingwith the transmembrane domain and including the rest of the C′terminalportion of the protein is set forth in amino acid residues 1060-1620 ofSEQ ID NO: 2.

The term “polypeptide” (or “amino acid sequence” or “protein”) refers toa polymer formed from the linking, in a defined order, of preferably,α-amino acids, D-, L-amino acids, and combinations thereof. The linkbetween one amino acid residue and the next is referred to as an amidebond or a peptide bond. Non-limiting examples of polypeptides include anoligopeptide, peptide, or protein sequence, and fragments or portionsthereof, and naturally occurring or synthetic molecules (e.g., peptidesynthesized by artificial means). Polypeptides also include derivatizedmolecules such as glycoproteins and lipoproteins as well as lowermolecular weight polypeptides. “Amino acid sequence” and like terms,such as “polypeptide” or “protein”, are not meant to limit the indicatedamino acid sequence to the complete, naturally-occurring amino acidsequence associated with the recited polypeptide molecule. Accordingly,the term “polypeptide” also includes variants of the recited polypeptidethat do not vary significantly from the structure or function of therecited polypeptide. If such differences in sequence are contemplated,it should be remembered that there will be critical areas on the proteinwhich determine activity (e.g. the kinase domain of ALK polypeptide). Ingeneral, it is possible to replace residues that form the tertiarystructure, provided that residues performing a similar function areused. In other instances, the type of residue may be completelyunimportant if the alteration occurs at a non-critical region of theprotein.

As used herein, the term “biological sample” refers to saliva, cells,mucous, tears, blood, serum, lymph/interstitial fluids, buccal cells,mucosal cells, cerebrospinal fluid, semen, feces, plasma, urine, marrow,a suspension of cells, or a suspension of cells and viruses or extractsor any of the foregoing, and may comprise a cell, chromosomes isolatedfrom a cell (e.g., a spread of metaphase chromosomes), genomic DNA (insolution or bound to a solid support such as for Southern analysis), RNA(in solution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support) obtainable from anymammal (e.g., a human), such as a normal mammal or a mammal having orsuspected of having kidney cancer. In some embodiments, a biologicalsample is mammalian (e.g., human) and is a biopsy sample or a bloodsample including a circulating tumor cell. Biological samples useful inthe practice of the methods of the invention may be obtained from anymammal.

Any biological sample comprising cells (or extracts of cells) from amammalian cancer is suitable for use in the methods of the invention. Inone embodiment, the biological sample comprises cells obtained from atumor biopsy. The biopsy may be obtained, according to standard clinicaltechniques, from primary tumors occurring in an organ of a mammal, or bysecondary tumors that have metastasized in other tissues. In anotherembodiment, the biological sample comprises cells obtained from a fineneedle aspirate taken from a tumor, and techniques for obtaining suchaspirates are well known in the art (see Cristallini et al., Acta Cytol.36(3): 416-22 (1992)).

In some embodiments, the biological sample comprises circulating tumorcells. Circulating tumor cells (“CTCs”) may be purified, for example,using the kits and reagents sold under the trademarks Vita-Assays™,Vita-Cap™, and CellSearch® (commercially available from Vitatex, LLC (aJohnson and Johnson corporation). Other methods for isolating CTCs aredescribed (see, for example, PCT Publication No. WO/2002/020825,Cristofanilli et al., New Engl. J. of Med. 351 (8):781-791 (2004), andAdams et al., J. Amer. Chem. Soc. 130(27): 8633-8641 (July 2008)). In aparticular embodiment, a circulating tumor cell (“CTC”) may be isolatedand identified as having originated from the kidney.

Accordingly, the invention provides a method for isolating a CTC, andthen screening the CTC one or more assay formats to identify thepresence of a polypeptide with ALK kinase activity a polynucleotideencoding the same in the CTC. Some non-limiting assay formats includeWestern blotting analysis, flow-cytometry (FC), immuno-histochemistry(IHC), immuno-fluorescence (IF), in situ hybridization (ISH),fluorescence in situ hybridization (FISH), and polymerase chain reaction(PCR). A CTC from a patient that is identified as comprising apolypeptide with ALK kinase activity or polynucleotide encoding the samemay indicate that the patient's originating cancer (e.g., a kidneycancer such as an squamous kidney cancer cell or a granular kidneycancer cell) is likely to respond to a composition comprising at leastone ALK kinase-inhibiting therapeutic.

Biological samples useful in the practice of the methods of theinvention may be obtained from any mammal in which a cancer driven bypolypeptide with ALK kinase activity is or might be present ordeveloping.

In various embodiments, the mammal (e.g., from which the mammaliankidney cancer or suspected mammalian kidney cancer originates) is ahuman, and the human may be a candidate for an ALK-inhibitingtherapeutic for the treatment of cancer driven by a polypeptide with ALKkinase activity. The human candidate may be a patient currently beingtreated with, or considered for treatment with, an ALK-inhibitingtherapeutic or other kinase-inhibiting therapeutic (e.g., Tarceva®). Inanother embodiment, the mammal is large animal, such as a horse or cow,while in other embodiments, the mammal is a small animal, such as a dogor cat, all of which are known to develop kidney cancer.

Any biological sample comprising cells (or extracts of cells) from amammalian cancer is suitable for use in the methods of the invention. Inone embodiment, the biological sample comprises cells obtained from atumor biopsy. The biopsy may be obtained, according to standard clinicaltechniques, from primary tumors occurring in an organ of a mammal, or bysecondary tumors that have metastasized in other tissues. In anotherembodiment, the biological sample comprises cells obtained from a fineneedle aspirate taken from a tumor by methods well known in the art (seeCristallini et al., Acta Cytol. 36(3): 416-22 (1992)).

Cellular extracts of the foregoing biological samples may be prepared,either crude or partially (or entirely) purified, in accordance withstandard techniques, and used in the various methods of the invention.Alternatively, biological samples comprising whole cells may be utilizedin assay formats such as immunohistochemistry (IHC), flow cytometry(FC), and immunofluorescence (IF), as further described herein. Suchwhole-cell assays are advantageous in that they minimize manipulation ofthe tumor cell sample and thus reduce the risks of altering the in vivosignaling/activation state of the cells and/or introducing artifactsignals. Whole cell assays are also advantageous because theycharacterize expression and signaling only in tumor cells, rather than amixture of tumor and normal cells.

As used herein, by “polypeptide having ALK kinase activity” is meant anyprotein or polypeptide (or fusion or fragment thereof) that retains thesignaling properties of a full length ALK protein (i.e., retains ALKkinase activity). Thus, polypeptides having ALK kinase activity include,without limitation, full-length ALK protein, portions of ALK comprisingthe kinase domain of ALK protein, truncated forms of ALK which retainALK kinase activity (e.g., a truncated ALK polypeptide comprising theALK kinase domain without the extracellular or transmembrane domain offull-length ALK) and all other ALK polypeptides, which may or may not befused with other polypeptides, that retain their ALK biological activityand/or tyrosine kinase activity. ALK may be derived from any species,such as mammalian, including bovine, ovine, porcine, murine, equine, andhuman, and may be derived from any source whether natural, synthetic,semi-synthetic, or recombinant. The human full length ALK protein is setforth in SEQ ID NO: 2. Persons of skill in the art would be readily ableto determine corresponding sequences in non-human mammalian ALKhomologues.

In various embodiments of all of the aspects of the invention, thepolypeptide with ALK kinase activity is aberrantly expressed full-lengthALK protein.

By “aberrantly expressed full-length ALK polypeptide” is meant that fulllength ALK is expressed in a cell of the kidney or kidney tissue of acancer or suspected cancer patient where, in the same cell or tissuetype of a normal individual, full-length ALK is not expressed. Suchaberrant expression may be due to, for example, mutations in regulatorysequences (such the promoter, enhancer, or intronic genomic sequences)operably linked to exons encoding amino acids of full-length ALKpolypeptide which result in aberrant expression of full-length ALKpolypeptide in the cell bearing the mutation. Numerous examples ofaberrantly expressed ALK kinase have been found in other cancers. Forexample, point mutations within the kinase domain have been found inneuroblastoma and overexpression of ALK has been found in numerouscancers (including, e.g., retinoblastoma, breast cancer, and melanoma).See review in Palmer et al., Biochem. J. 420(3): 345-361 (May 2009),herein incorporated by reference in its entirety. Aberrant expression(e.g., overexpression) of full length ALK polypeptide in a cancer (e.g.,a kidney cancer) may be the result of amplification of the ALK gene inthe cancer cell's genome.

In various embodiments, the polypeptide with ALK kinase activity is anALK fusion polypeptide comprising at least a portion of a first fusionmember and at least a portion of a second fusion member, wherein thesecond fusion member is an ALK protein comprises an ALK kinase domain.

The term “ALK fusion polypeptide” refers to a portion or fragment of theALK protein fused to at least a portion or fragment of another protein.In some embodiments, the portion of ALK protein present in an ALK fusionpolypeptide comprises the kinase domain of full-length ALK protein. Insome embodiments, the portions of the ALK present in an ALK fusionpolypeptide comprise amino acids encoded by exon 20 onward of anALK-encoding gene. In some embodiments, an ALK fusion polypeptidecomprises the amino acid sequence set forth in SEQ ID NO: 3. In someembodiments, an ALK fusion polypeptide comprises the amino acid sequenceset forth in SEQ ID NO: 4. In some embodiments, an ALK fusionpolypeptide comprises the amino acid sequence set forth in SEQ ID NO:15. An ALK fusion polypeptide often results from a chromosomaltranslocation or inversion. Non-limiting examples of ALK fusionpolypeptides include VCL-ALK, EML4-ALK, NPM-ALK, TPM3-ALK, TFG-ALK,ATIC-ALK, CLTC-ALK, MSN-ALK, TPM4-ALK, ALO17-ALK, RANBP2-ALK, MYH9-ALK,CARS-ALK, SEC31L1-ALK, and KIF5B-ALK (see, e.g., Debelenko et al.,Modern Pathology 24: 430-442, 2011; Marino-Enriquez et al., Genes,Chromosome, and Cancer 50(3): 146-153, 2011; Palmer et al., Biochem. J.420(3): 345-361, 2009 (and the articles cited therein), Rikova et al.,Cell 131: 1190-1203, 2007; Soda et al., Nature 448: 561-566, 2007;Morris et al., Science 263: 1281-1284, 1994; Du et al., J. Mol. Med 84:863-875, 2007; Panagopoulos et al., Int. J. Cancer 118: 1181-1186, 2006;Cools et al., Genes Chromosomes Cancer 34: 354-362, 2002; Debelenko etal., Lab. Invest. 83: 1255-1265, 2003; Ma et al., Genes ChromosomesCancer 37: 98-105, 2003; Lawrence et al., Am. J. Pathol. 157: 377-384,2000; Hernandez et al., Blood 94: 3265-3268, 1999; Hernandez et al., AmJ Pathol 160: 1487-1494, 2002; Takeuchi K., Clin Cancer Res.15(9):3143-3149, 2009; Tort et al., Lab. Invest. 81: 419-426, 2001;Trinei et al., Cancer Res. 60: 793-798, 2000; Colleoni et al., Am JPathol. 156 (3): 781-9, 2000; Shiota et al, Blood 86: 1954-1960, 1995;Kuefer et al., Blood 90: 2901-2910, 1997; Shiota et al., Oncogene 9:1567-1574, 1994; Touriol et al., Blood 95: 3204-3207, 2000; and Pulfordet al., Journal of Cellular Physiology, 199:330-358, 2004. Some of theseALK fusions have multiple variants, all of which are considered ALKfusions and, thus, are included in the definition of mutant ALK of theinvention. For example, there are multiple variants of TFG-ALK (see,e.g., Hernandez et al., Amer. J. Pathol. 160: 1487-1494, 2002) and atleast nine known variants of EML4-ALK (see, e.g., Horn et al., J. ofClinical Oncology 27(26): 4232-4235, 2009, U.S. Pat. Nos. 7,728,120;7,700,339 and EP Patent No. 1914 240). Moreover, a method foridentifying a protein as a fusion partner for ALK using ALK antibodieshas been reported (Elenitoba-Johnson et al., Proc. Natl. Acad. Sci. 103:7402-7407, 2006).

It should be noted that in all of the ALK fusion proteins describedherein, the amino acid at the fusion junction (regardless of thenumbering) may appear in either full-length protein member of thefusion, or the amino acid, being created by a codon with nucleotidesfrom fused exons and/or of both protein members, may be unique to thefusion polypeptide and not appear in either full-length protein memberof the fusion.

As used herein, a “portion” or “fragment” means a sequence fragment lessthan the whole sequence. For example, a 50 nucleotide sequence is aportion of a 100 nucleotide long sequence. Similarly, a 50 amino acidresidue long sequence is a portion of a 100 amino acid long sequence. Insome embodiments, a nucleic acid fragment or portion comprises at least20 nucleotides, or at least 30 nucleotides, or at least 45 nucleotides,or at least 60 nucleotides, or at least 70 nucleotides, or at least 90nucleotides. In some embodiments, a polypeptide fragment or portioncomprises at least 6 amino acid residues, or at least 10 amino acidresidues, or at least 20 amino acid residues, or at least 30 amino acidresidues, or at least 45 amino acid residues, or at least 60 amino acidresidues, or at least 70 amino acid residues, or at least 90 amino acidresidues.

In one embodiment, the ALK fusion polypeptide contains the complete ALKtyrosine kinase domain located at amino acids 1116-1392 of ALK (SEQ IDNO: 3). In another embodiment, the ALK fusion polypeptide contains thecomplete intracytoplasmic domain located at amino acids 1060-1620 of ALK(SEQ ID NO: 4). In another embodiment, the ALK fusion polypeptidecontains amino acid residues 1504-1507 (SEQ ID NO:5) of ALK making upthe phosphotyrosine-binding site of the C-terminal domain of ALK. In yetanother embodiment, the ALK fusion polypeptide contains amino acidresidues 1603-1606 (SEQ ID NO:6) of ALK representing the interactionsite for the phosphotyrosine-dependent binding of the substratephospholipase C-γ (PLC-γ).

Because ALK is not known to be expressed in normal kidney cells, thepresence or activity of a polypeptide with ALK kinase activity inmammalian kidney cancer or suspected mammalian kidney cancer is, inaccordance with the invention, identified as a mammalian kidney canceror suspected mammalian kidney cancer belonging to a subset of kidneycancers driven by ALK kinase activity.

As used herein, by “drive” or “driven” is meant that a mammalian kidneycancer or suspected mammalian kidney cancer has gained its cancerousstate because of the presence within the cells of the mammalian kidneycancer or suspected mammalian kidney cancer of a polypeptide with ALKkinase activity. Such a presence may be detected by detecting thepresence of a polynucleotide encoding the polypeptide with ALK kinaseactivity (e.g., detecting a gene translocation involving the ALK geneand another gene or detecting a mutation in the promoter of the ALK genethat would result in aberrant expression of full-length ALKpolypeptide), detecting the polypeptide with ALK kinase activity, or bydetecting ALK kinase activity of the polypeptide with ALK kinaseactivity. In other words, the presence of a polypeptide with ALK kinaseactivity stimulates or is the causative agent of the cancerous state ofthe mammalian kidney cancer or suspected mammalian kidney cancer.

As used herein, by “cancer” or “cancerous” is meant a cell that showsabnormal growth as compared to a normal (i.e., non-cancerous) cell ofthe same cell type. For example, a cancerous cell may be hyperplastic,anaplastic, metastatic, or benign (but shows abnormal growth). Acancerous cell may also show lack of contact inhibition where a normalcell of that same cell type shows contact inhibition.

In various embodiments of the invention, the activity of a polypeptidewith ALK kinase activity is detected using as a detection molecule areagent that detects ALK kinase activity.

Numerous reagents can be employed that detect ALK kinase activity. Forexample, substrates of ALK kinase are known (see, e.g., Donella-Deana etal., Biochemistry 44(23):8533-8542, 2005; Simonitsch et al., FASEB J15:1416-1418, 2001; and the Poly(Glu:Tyr)4:1 substrate commerciallyavailable from Sigma-Aldrich (St. Louis, Mo.; Catalog No. P-0275)).These substrates can be added to standard in vitro kinase assays andtheir phosphorylation on tyrosine determined (using, e.g., aphosphotyrosine-specific antibody such as the 4G10 antibody commerciallyavailable from Millipore, Bedford, Mass. or the P-Tyr-100 antibody(Catalog No. 9411) commercially available from Cell SignalingTechnology, Inc., Danvers, Mass.

Note that because ALK autophosphorylates when its kinase activity isactive (see Perez-Pinera et al., Journal of Biological Chemistry 282:28683-28690, 207), the reagent that detects ALK kinase activity mayitself be a phosphotyrosine-specific antibody that binds totyrosine-phosphorylated ALK protein.

In some embodiments, ALK kinase activity is detected in an in vitrokinase assay format. In vitro kinase assays have been well known formany years (see, e.g., Ausubel et al., supra; Hernandez et al., Blood94: 3265-3268, 1999). Indeed, in vitro kinase services for detecting ALKkinase activity are commercially available (from ProQinase GmbH,Freiburg, Germany).

In some embodiments, the method for detecting the presence and/oractivity of a polypeptide with ALK kinase activity in a biologicalsample from a mammalian kidney cancer or suspected mammalian kidneycancer comprises the steps of: (a) obtaining a biological sample from amammalian kidney cancer or suspected mammalian kidney cancer and (b)contacting the biological sample with a reagent that detects apolypeptide with ALK kinase activity, wherein detection of saidpolypeptide in said biological sample indicates said polypeptide withALK kinase activity is present in said biological sample.

In various embodiments of the invention, the expression of a polypeptidewith ALK kinase activity is detected using as a detection molecule areagent that detects a polypeptide with ALK kinase activity. In someembodiments, the reagent specifically binds to a polypeptide with ALKkinase activity.

By “specifically binding” or “specifically binds” means that a reagentthat may be used in the various methods of the invention (e.g., anantibody or AQUA peptide) interacts with its target molecule (e.g., apolypeptide with ALK kinase activity such as a full-length ALKpolypeptide or a ALK fusion polypeptide), where the interaction isdependent upon the presence of a particular structure (e.g., theantigenic determinant or epitope on the polypeptide or the nucleotidesequence of the polynucleotide); in other words, the reagent isrecognizing and binding to a specific polypeptide or polynucleotidestructure rather than to all polypeptides or polynucleotides in general.By “binding fragment thereof” means a fragment or portion of a reagentthat specifically binds the target molecule (e.g., an Fab fragment of anantibody).

A reagent that specifically binds to the target molecule may be referredto as a target-specific reagent or an anti-target reagent. For example,an antibody that specifically binds to a NPM-ALK polypeptide may bereferred to as a NPM-ALK-specific antibody or an anti-NPM-ALK antibody.Likewise, an antibody that specifically binds to the full length ALKpolypeptide may be referred to as an ALK-specific antibody or ananti-ALK antibody.

In some embodiments, a reagent that specifically binds its targetmolecule has a binding affinity (K_(D)) for its target molecule (e.g.,an ALK fusion polypeptide) of 1×10⁻⁶ M or less. In some embodiments, areagent that specifically binds to its target molecule binds to itstarget molecule with a K_(D) of 1×10⁻⁷ M or less, or a K_(D)Of 1×10⁻⁸ Mor less, or a K_(D) of 1×10⁻⁹ M or less, or a K_(D) of 1×10⁻¹⁰ M orless, of a K_(D) of 1×10⁻¹¹ M or less, of a K_(D) of 1×10⁻¹² M or less.In certain embodiments, a reagent that specifically binds to its targetmolecule binds to its target molecule with a K_(D) of 1 pM to 500 pM, orbetween 500 pM to 1 pM, or between 1 pM to 100 nM, or between 100 mM to10 nM.

In some embodiments, a reagent that specifically binds to a polypeptidewith ALK kinase activity is a heavy-isotope labeled peptide (i.e., anAQUA peptide). Such an AQUA peptide may be suitable for the absolutequantification of an expressed polypeptide with ALK kinase activity in abiological sample. As used herein, the term “heavy-isotope labeledpeptide” is used interchangeably with “AQUA peptide”. The production anduse of AQUA peptides for the absolute quantification or detection ofproteins (AQUA) in complex mixtures has been described. See PCTPublication No. WO/03016861 and also Gerber et al., Proc. Natl. Acad.Sci. U.S.A. 100: 6940-5 (2003). The term “specifically detects” withrespect to such an AQUA peptide means the peptide will only detect andquantify polypeptides and proteins that contain the AQUA peptidesequence and will not substantially detect polypeptides and proteinsthat do not contain the AQUA peptide sequence.

AQUA internal peptide standards (heavy-isotope labeled peptides) maydesirably be produced to detect any quantify any unique site (e.g., thefusion junction within an ALK fusion polypeptide) within a polypeptidewith ALK kinase activity.

The AQUA methodology employs the introduction of a known quantity of atleast one heavy-isotope labeled peptide standard (which has a uniquesignature detectable by LC-SRM chromatography) into a digestedbiological sample in order to determine, by comparison to the peptidestandard, the absolute quantity of a peptide with the same sequence andprotein modification in the biological sample. Briefly, the AQUAmethodology has two stages: peptide internal standard selection andvalidation and method development; and implementation using validatedpeptide internal standards to detect and quantify a target protein insample. The method is a powerful technique for detecting and quantifyinga given peptide/protein within a complex biological mixture, such as acell lysate, and may be employed, e.g., to quantify change in proteinphosphorylation as a result of drug treatment, or to quantifydifferences in the level of a protein in different biological states.

Generally, to develop a suitable internal standard, a particular peptide(or modified peptide) within a target protein sequence is chosen basedon its amino acid sequence and the particular protease to be used todigest. The peptide is then generated by solid-phase peptide synthesissuch that one residue is replaced with that same residue containingstable isotopes (¹³C, ¹⁵N). The result is a peptide that is chemicallyidentical to its native counterpart formed by proteolysis, but is easilydistinguishable by MS via a 7-Da mass shift. The newly synthesized AQUAinternal standard peptide is then evaluated by LC-MS/MS. This processprovides qualitative information about peptide retention byreverse-phase chromatography, ionization efficiency, and fragmentationvia collision-induced dissociation. Informative and abundant fragmentions for sets of native and internal standard peptides are chosen andthen specifically monitored in rapid succession as a function ofchromatographic retention to form a selected reaction monitoring(LC-SRM) method based on the unique profile of the peptide standard.

The second stage of the AQUA strategy is its implementation to measurethe amount of a protein or modified protein from complex mixtures. Wholecell lysates are typically fractionated by SDS-PAGE gel electrophoresis,and regions of the gel consistent with protein migration are excised.This process is followed by in-gel proteolysis in the presence of theAQUA peptides and LC-SRM analysis. (See Gerber et al., supra.) AQUApeptides are spiked in to the complex peptide mixture obtained bydigestion of the whole cell lysate with a proteolytic enzyme andsubjected to immunoaffinity purification as described above. Theretention time and fragmentation pattern of the native peptide formed bydigestion (e.g., trypsinization) is identical to that of the AQUAinternal standard peptide determined previously; thus, LC-MS/MS analysisusing an SRM experiment results in the highly specific and sensitivemeasurement of both internal standard and analyte directly fromextremely complex peptide mixtures.

Since an absolute amount of the AQUA peptide is added (e.g., 250 fmol),the ratio of the areas under the curve can be used to determine theprecise expression levels of a protein or phosphorylated form of aprotein in the original cell lysate. In addition, the internal standardis present during in-gel digestion as native peptides are formed, suchthat peptide extraction efficiency from gel pieces, absolute lossesduring sample handling (including vacuum centrifugation), andvariability during introduction into the LC-MS system do not affect thedetermined ratio of native and AQUA peptide abundances.

An AQUA peptide standard is developed for a known sequence previouslyidentified by the IAP-LC-MS/MS method within in a target protein. If thesite is modified, one AQUA peptide incorporating the modified form ofthe particular residue within the site may be developed, and a secondAQUA peptide incorporating the unmodified form of the residue developed.In this way, the two standards may be used to detect and quantify boththe modified an unmodified forms of the site in a biological sample.

Peptide internal standards may also be generated by examining theprimary amino acid sequence of a protein and determining the boundariesof peptides produced by protease cleavage. Alternatively, a protein mayactually be digested with a protease and a particular peptide fragmentproduced can then sequenced. Suitable proteases include, but are notlimited to, serine proteases (e.g. trypsin, hepsin), metallo proteases(e.g., PUMP1), chymotrypsin, cathepsin, pepsin, thermolysin,carboxypeptidases, etc.

A peptide sequence within a target protein is selected according to oneor more criteria to optimize the use of the peptide as an internalstandard. Preferably, the size of the peptide is selected to minimizethe chances that the peptide sequence will be repeated elsewhere inother non-target proteins. Thus, a peptide is preferably at least about6 amino acids. The size of the peptide is also optimized to maximizeionization frequency. Thus, in some embodiments, the peptide is notlonger than about 20 amino acids. In some embodiments, the peptide isbetween about 7 to 15 amino acids in length. A peptide sequence is alsoselected that is not likely to be chemically reactive during massspectrometry, thus sequences comprising cysteine, tryptophan, ormethionine are avoided.

A peptide sequence that does not include a modified region of the targetregion may be selected so that the peptide internal standard can be usedto determine the quantity of all forms of the protein. Alternatively, apeptide internal standard encompassing a modified amino acid may bedesirable to detect and quantify only the modified form of the targetprotein. Peptide standards for both modified and unmodified regions canbe used together, to determine the extent of a modification in aparticular sample (i.e. to determine what fraction of the total amountof protein is represented by the modified form). For example, peptidestandards for both the phosphorylated and unphosphorylated form of aprotein known to be phosphorylated at a particular site can be used toquantify the amount of phosphorylated form in a sample.

The peptide is labeled using one or more labeled amino acids (i.e., thelabel is an actual part of the peptide) or less preferably, labels maybe attached after synthesis according to standard methods. Preferably,the label is a mass-altering label selected based on the followingconsiderations: The mass should be unique to shift fragments massesproduced by MS analysis to regions of the spectrum with low background;the ion mass signature component is the portion of the labeling moietythat preferably exhibits a unique ion mass signature in MS analysis; thesum of the masses of the constituent atoms of the label is preferablyuniquely different than the fragments of all the possible amino acids.As a result, the labeled amino acids and peptides are readilydistinguished from unlabeled ones by the ion/mass pattern in theresulting mass spectrum. Preferably, the ion mass signature componentimparts a mass to a protein fragment that does not match the residuemass for any of the 20 natural amino acids.

The label should be robust under the fragmentation conditions of MS andnot undergo unfavorable fragmentation. Labeling chemistry should beefficient under a range of conditions, particularly denaturingconditions, and the labeled tag preferably remains soluble in the MSbuffer system of choice. The label preferably does not suppress theionization efficiency of the protein and is not chemically reactive. Thelabel may contain a mixture of two or more isotopically distinct speciesto generate a unique mass spectrometric pattern at each labeled fragmentposition. Stable isotopes, such as ²H, ¹³C, ¹⁵N, ¹⁷O, ¹⁸O, or ³⁴S, aresome non-limiting labels. Pairs of peptide internal standards thatincorporate a different isotope label may also be prepared. Non-limitingamino acid residues into which a heavy isotope label may be incorporatedinclude leucine, proline, valine, and phenylalanine.

Peptide internal standards are characterized according to theirmass-to-charge (m/z) ratio, and preferably, also according to theirretention time on a chromatographic column (e.g., an HPLC column).Internal standards that co-elute with unlabeled peptides of identicalsequence are selected as optimal internal standards. The internalstandard is then analyzed by fragmenting the peptide by any suitablemeans, for example by collision-induced dissociation (CID) using, e.g.,argon or helium as a collision gas. The fragments are then analyzed, forexample by multi-stage mass spectrometry (MS^(n)) to obtain a fragmention spectrum, to obtain a peptide fragmentation signature. Preferably,peptide fragments have significant differences in m/z ratios to enablepeaks corresponding to each fragment to be well separated, and asignature is that is unique for the target peptide is obtained. If asuitable fragment signature is not obtained at the first stage,additional stages of MS are performed until a unique signature isobtained.

Fragment ions in the MS/MS and MS³ spectra are typically highly specificfor the peptide of interest, and, in conjunction with LC methods, allowa highly selective means of detecting and quantifying a targetpeptide/protein in a complex protein mixture, such as a cell lysate,containing many thousands or tens of thousands of proteins. Anybiological sample potentially containing a target protein/peptide ofinterest may be assayed. Crude or partially purified cell extracts arepreferably employed. Generally, the sample has at least 0.01 mg ofprotein, typically a concentration of 0.1-10 mg/mL, and may be adjustedto a desired buffer concentration and pH.

A known amount of a labeled peptide internal standard, preferably about10 femtomoles, corresponding to a target protein to bedetected/quantified is then added to a biological sample, such as a celllysate. The spiked sample is then digested with one or more protease(s)for a suitable time period to allow digestion. A separation is thenperformed (e.g. by HPLC, reverse-phase HPLC, capillary electrophoresis,ion exchange chromatography, etc.) to isolate the labeled internalstandard and its corresponding target peptide from other peptides in thesample. Microcapillary LC is a one non-limiting method.

Each isolated peptide is then examined by monitoring of a selectedreaction in the MS. This involves using the prior knowledge gained bythe characterization of the peptide internal standard and then requiringthe MS to continuously monitor a specific ion in the MS/MS or MS^(n)spectrum for both the peptide of interest and the internal standard.After elution, the area under the curve (AUC) for both peptide standardand target peptide peaks are calculated. The ratio of the two areasprovides the absolute quantification that can be normalized for thenumber of cells used in the analysis and the protein's molecular weight,to provide the precise number of copies of the protein per cell. Furtherdetails of the AQUA methodology are described in PCT Publication No.WO/03016861 and Gerber et al. supra.

In some embodiments, a reagent that specifically binds to a polypeptidewith ALK kinase activity is an antibody. In some embodiments, theantibody is specific for (i.e., specifically binds to) full length ALKpolypeptide. In some embodiments, the antibody does not cross-react with(i.e., does not specifically bind to) c-Met. In some embodiments, theantibody is specific for the kinase domain of ALK. In some embodiments,the antibody is specific for the extracellular domain of ALK. In someembodiments, where the polypeptide with ALK kinase activity is an ALKfusion polypeptide, the antibody specifically binds to the portion ofALK polypeptide that is fused to the portion of ALK kinase present inthe ALK fusion polypeptide. For example, if the fusion is EML4-ALK (the796 variant), the antibody specifically binds to the portion of the ALKprotein (e.g., the ALK kinase domain) present in the fusion. In someembodiments, where the polypeptide with ALK kinase activity is an ALKfusion polypeptide, the antibody specifically binds to the portion ofthe fusion partner that is fused to the portion of ALK kinase present inthe ALK fusion polypeptide. For example, if the fusion is EML4-ALK (the796 variant), the antibody specifically binds to the N-terminus of theEML4 protein present in the fusion.

The term “antibody” or “antibodies” refers to all types ofimmunoglobulins, including IgG, IgM, IgA, IgD, and IgE, includingbinding fragments thereof (i.e., fragments of an antibody that arecapable of specifically binding to the antibody's target molecule, suchas F_(ab), and F(ab′)₂ fragments), as well as recombinant, humanized,polyclonal, and monoclonal antibodies and/or binding fragments thereof.Antibodies of the invention can be derived from any species of animal,such as from a mammal. Non-limiting exemplary natural antibodies includeantibodies derived from human, chicken, goats, and rodents (e.g., rats,mice, hamsters and rabbits), including transgenic rodents geneticallyengineered to produce human antibodies (see, e.g., Lonberg et al.,WO93/12227; U.S. Pat. No. 5,545,806; and Kucherlapati, et al.,WO91/10741; U.S. Pat. No. 6,150,584, which are herein incorporated byreference in their entirety). Antibodies of the invention may be also bechimeric antibodies. See, e.g., M. Walker et al., Molec. Immunol. 26:403-11 (1989); Morrision et al., Proc. Nat'l. Acad. Sci. 81: 6851(1984); Neuberger et al., Nature 312: 604 (1984)). The antibodies may berecombinant monoclonal antibodies produced according to known methods(see, e.g., U.S. Pat. Nos. 4,474,893; 4,816,567; 7,485,291, and USPatent Publication No. 20110045534). The antibodies may also bechemically constructed specific antibodies made according to the methoddisclosed in U.S. Pat. No. 4,676,980.

Natural antibodies are the antibodies produced by a host animal, howeverthe invention contemplates also genetically altered antibodies whereinthe amino acid sequence has been varied from that of a native antibody.Because of the relevance of recombinant DNA techniques to thisapplication, one need not be confined to the sequences of amino acidsfound in natural antibodies; antibodies can be redesigned to obtaindesired characteristics. The possible variations are many and range fromthe changing of just one or a few amino acids to the complete redesignof, for example, the variable or constant region. Changes in theconstant region will, in general, be made in order to improve or altercharacteristics, such as complement fixation, interaction with membranesand other effector functions. Changes in the variable region will bemade in order to improve the antigen binding characteristics. The term“humanized antibody”, as used herein, refers to antibody molecules inwhich amino acids have been replaced in the non-antigen binding regionsin order to more closely resemble a human antibody, while stillretaining the original binding ability. Other antibodies specificallycontemplated are oligoclonal antibodies. As used herein, the phrase“oligoclonal antibodies” refers to a predetermined mixture of distinctmonoclonal antibodies. See, e.g., PCT publication WO 95/20401; U.S. Pat.Nos. 5,789,208 and 6,335,163. In one embodiment, oligoclonal antibodiesconsisting of a predetermined mixture of antibodies against one or moreepitopes are generated in a single cell. In other embodiments,oligoclonal antibodies comprise a plurality of heavy chains capable ofpairing with a common light chain to generate antibodies with multiplespecificities (e.g., PCT publication WO 04/009618). Oligoclonalantibodies are particularly useful when it is desired to target multipleepitopes on a single target molecule. In view of the assays and epitopesdisclosed herein, those skilled in the art can generate or selectantibodies or mixtures of antibodies that are applicable for an intendedpurpose and desired need.

Recombinant antibodies are also included in the present invention. Theserecombinant antibodies have the same amino acid sequence as the naturalantibodies or have altered amino acid sequences of the naturalantibodies. They can be made in any expression systems including bothprokaryotic and eukaryotic expression systems or using phage displaymethods (see, e.g., Dower et al., WO91/17271 and McCafferty et al.,WO92/01047; U.S. Pat. No. 5,969,108, which are herein incorporated byreference in their entirety). Antibodies can be engineered in numerousways. They can be made as single-chain antibodies (including smallmodular immunopharmaceuticals or SMIPs™), Fab and F(ab′)₂ fragments,etc. Antibodies can be humanized, chimerized, deimmunized, or fullyhuman. Numerous publications set forth the many types of antibodies andthe methods of engineering such antibodies. For example, see U.S. Pat.Nos. 6,355,245; 6,180,370; 5,693,762; 6,407,213; 6,548,640; 5,565,332;5,225,539; 6,103,889; and 5,260,203. The genetically altered antibodiesof the invention may be functionally equivalent to the above-mentionednatural antibodies. In certain embodiments, modified antibodies of theinvention provide improved stability or/and therapeutic efficacy.

Non-limiting examples of modified antibodies include those withconservative substitutions of amino acid residues, and one or moredeletions or additions of amino acids that do not significantlydeleteriously alter the antigen binding utility. Substitutions can rangefrom changing or modifying one or more amino acid residues to completeredesign of a region as long as the therapeutic utility is maintained.Antibodies of the invention can be modified post-translationally (e.g.,acetylation, and/or phosphorylation) or can be modified synthetically(e.g., the attachment of a labeling group). Antibodies with engineeredor variant constant or Fc regions can be useful in modulating effectorfunctions, such as, for example, antigen-dependent cytotoxicity (ADCC)and complement-dependent cytotoxicity (CDC). Such antibodies withengineered or variant constant or Fc regions may be useful in instanceswhere a parent singling protein is expressed in normal tissue; variantantibodies without effector function in these instances may elicit thedesired therapeutic response while not damaging normal tissue.Accordingly, certain aspects and methods of the present disclosurerelate to antibodies with altered effector functions that comprise oneor more amino acid substitutions, insertions, and/or deletions. The term“biologically active” refers to a protein having structural, regulatory,or biochemical functions of a naturally occurring molecule. Likewise,“immunologically active” refers to the capability of the natural,recombinant, or synthetic full-length ALK protein or ALK fusionpolypeptide (e.g., a FN1-ALK fusion polypeptide or an FN1-tmALK fusionpolypeptide of the invention), or any oligopeptide thereof, to induce aspecific immune response in appropriate animals or cells and to bindwith specific antibodies.

Also within the invention are antibody molecules with fewer than 4chains, including single chain antibodies, Camelid antibodies and thelike and components of an antibody, including a heavy chain or a lightchain. In some embodiments an immunoglobulin chain may comprise in orderfrom 5′ to 3′, a variable region and a constant region. The variableregion may comprise three complementarity determining regions (CDRs),with interspersed framework (FR) regions for a structure FR1, CDR1, FR2,CDR2, FR3, CDR3 and FR4. Also within the invention are heavy or lightchain variable regions, framework regions and CDRs. An antibody of theinvention may comprise a heavy chain constant region that comprises someor all of a CH1 region, hinge, CH2 and CH3 region.

One non-limiting epitopic site of a ALK fusion polypeptide-specificantibody of the invention is a peptide fragment consisting essentiallyof about 11 to 17 amino acids of a fusion polypeptide sequence, whichfragment encompasses the fusion junction between the ALK portion and theportion of the second fusion partner present in the ALK fusionpolypeptide. It will be appreciated that antibodies that specificallybinding shorter or longer peptides/epitopes encompassing the fusionjunction of an ALK fusion polypeptide are within the scope of thepresent invention.

The invention is not limited to use of antibodies, but includesequivalent molecules, such as protein binding domains or nucleic acidaptamers, which bind, in a fusion-protein or truncated-protein specificmanner, to essentially the same epitope to which a polypeptide withkinase activity-specific antibody useful in the methods of the inventionbinds. See, e.g., Neuberger et al., Nature 312: 604 (1984). Suchequivalent non-antibody reagents may be suitably employed in the methodsof the invention further described below.

Polyclonal antibodies useful in practicing the methods of the inventionmay be produced according to standard techniques by immunizing asuitable animal (e.g., rabbit, goat, etc.) with an antigen encompassinga desired epitope (e.g. the fusion junction between the ALK portion andthe portion of the second fusion partner present in the ALK fusionpolypeptide), collecting immune serum from the animal, and separatingthe polyclonal antibodies from the immune serum, and purifyingpolyclonal antibodies having the desired specificity, in accordance withknown procedures. The antigen may be a synthetic peptide antigencomprising the desired epitopic sequence, selected and constructed inaccordance with well-known techniques. See, e.g., ANTIBODIES: ALABORATORY MANUAL, Chapter 5, p. 75-76, Harlow & Lane Eds., Cold SpringHarbor Laboratory (1988); Czernik, Methods In Enzymology, 201: 264-283(1991); Merrifield, J. Ani. Chem. Soc. 85: 21-49 (1962)). Polyclonalantibodies produced as described herein may be screened and isolated asfurther described below.

Monoclonal antibodies may also be beneficially employed in the methodsof the invention, and may be produced in hybridoma cell lines accordingto the well-known technique of Kohler and Milstein. Nature 265: 495-97(1975); Kohler and Milstein, Eur. J. Immunol. 6: 511 (1976); see also,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al. Eds. (Wiley andSins, New York, N.Y. 1989 and yearly updates up to and including 2010).Monoclonal antibodies so produced are highly specific, and improve theselectivity and specificity of assay methods provided by the invention.For example, a solution containing the appropriate antigen (e.g. asynthetic peptide comprising the fusion junction of the ALK portion andthe portion of the second fusion partner present in the ALK fusionpolypeptide) may be injected into a mouse and, after a sufficient time(in keeping with conventional techniques), the mouse sacrificed andspleen cells obtained. The spleen cells are then immortalized by fusingthem with myeloma cells, typically in the presence of polyethyleneglycol, to produce hybridoma cells. Rabbit fusion hybridomas, forexample, may be produced as described in U.S. Pat. No. 5,675,063. Thehybridoma cells are then grown in a suitable selection media, such ashypoxanthine-aminopterin-thymidine (HAT), and the supernatant screenedfor monoclonal antibodies having the desired specificity, as describedbelow. The secreted antibody may be recovered from tissue culturesupernatant by conventional methods such as precipitation, ion exchangeor affinity chromatography, or the like.

Monoclonal Fab fragments may also be produced in Escherichia coli byrecombinant techniques known to those skilled in the art. See, e.g., W.Huse, Science 246: 1275-81 (1989); Mullinax et al., Proc. Nat'l Acad.Sci. 87: 8095 (1990). If monoclonal antibodies of one isotype aredesired for a particular application, particular isotypes can beprepared directly, by selecting from the initial fusion, or preparedsecondarily, from a parental hybridoma secreting a monoclonal antibodyof different isotype by using the sib selection technique to isolateclass-switch variants (Steplewski, et a., Proc. Nat'l. Acad. Sci., 82:8653 (1985); Spira et al., J. Immunol. Methods, 74: 307 (1984)). Theantigen combining site of the monoclonal antibody can be cloned by PCRand single-chain antibodies produced as phage-displayed recombinantantibodies or soluble antibodies in E. coli (see, e.g., ANTIBODYENGINEERING PROTOCOLS, 1995, Humana Press, Sudhir Paul editor.)

Further still, U.S. Pat. No. 5,194,392, Geysen (1990) describes ageneral method of detecting or determining the sequence of monomers(amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a “mimotope”) which is complementary to a particularparatope (antigen binding site) of an antibody of interest. Moregenerally, this method involves detecting or determining a sequence ofmonomers which is a topographical equivalent of a ligand which iscomplementary to the ligand binding site of a particular receptor ofinterest. Similarly, U.S. Pat. No. 5,480,971, Houghten et al. (1996)discloses linear C₁-C-alkyl peralkylated oligopeptides and sets andlibraries of such peptides, as well as methods for using sucholigopeptide sets and libraries for determining the sequence of aperalkylated oligopeptide that preferentially binds to an acceptormolecule of interest. Thus, non-peptide analogs of the epitope-bearingpeptides of the invention also can be made routinely by these methods.

Antibodies useful in the methods of the invention, whether polyclonal ormonoclonal, may be screened for epitope and fusion protein specificityaccording to standard techniques. See, e.g., Czernik et al., Methods inEnzynology, 201: 264-283 (1991). For example, the antibodies may bescreened against a peptide library by ELISA to ensure specificity forboth the desired antigen and, if desired, for reactivity only with thefull-length ALK protein, a particular ALK fusion polypeptide (e.g., anEML4-ALK (1059 amino acid variant) polypeptide), or fragments thereof ofthe invention. The antibodies may also be tested by Western blottingagainst cell preparations containing target protein to confirmreactivity with the only the desired target and to ensure no appreciablebinding to other proteins. The production, screening, and use of fusionprotein-specific antibodies is known to those of skill in the art, andhas been described. See, e.g., U.S. Patent Publication No. 20050214301.

Full-length ALK protein-specific and ALK fusion polypeptide-specificantibodies useful in the methods of the invention may exhibit somelimited cross-reactivity with similar epitopes in other proteins orpolypeptides, such as similar fusion polypeptides. This is notunexpected as most antibodies exhibit some degree of cross-reactivity,and anti-peptide antibodies will often cross-react with epitopes havinghigh homology or identity to the immunizing peptide. See, e.g., Czernik,supra. Cross-reactivity with other fusion proteins is readilycharacterized by Western blotting alongside markers of known molecularweight. Amino acid sequences of cross-reacting proteins may be examinedto identify sites highly homologous or identical to full length ALKprotein sequence or the ALK fusion polypeptide sequence to which theantibody binds. Undesirable cross-reactivity can be removed by negativeselection using antibody purification on peptide columns.

Polypeptide with ALK kinase activity-specific antibodies and ALK fusionpolypeptide-specific antibodies of the invention that are useful inpracticing the methods disclosed herein are ideally specific for humanfusion polypeptide, but are not limited only to binding the humanspecies, per se. The invention includes the production and use ofantibodies that also bind conserved and highly homologous or identicalepitopes in other mammalian species (e.g., mouse, rat, monkey). Highlyhomologous or identical sequences in other species can readily beidentified by standard sequence comparisons, such as using BLAST, withthe human ALK protein sequence (SEQ ID NO: 2.

Antibodies employed in the methods of the invention may be furthercharacterized by, and validated for, use in a particular assay format,for example flow cytometry (FC), immunohistochemistry (IHC), and/orimmunocytochemistry (ICC). The use of polypeptide with ALK kinaseactivity-specific antibodies in such methods is further describedherein. The antibodies described herein, used alone or in thebelow-described assays, may also be advantageously conjugated tofluorescent dyes (e.g. Alexa488, phycoerythrin), or labels such asquantum dots, for use in multi-parametric analyses along with othersignal transduction (phospho-AKT, phospho-Erk 1/2) and/or cell marker(cytokeratin) antibodies, as further described below.

In practicing the methods of the invention, the expression and/oractivity of a polypeptide with ALK kinase activity (e.g., a full-lengthALK polypeptide) in a given biological sample may also be advantageouslyexamined using antibodies specific for (i.e., that specifically bind to)full length ALK protein or antibodies specific for ALK fusionpolypeptides. For example, ALK-specific antibodies (i.e., antibodiesthat specifically bind full-length ALK) are commercially available (seeCELL SIGNALING TECHNOLOGY, INC., Danvers, Mass., Catalog Nos. 3333 and3791; Abcam, 2010 Catalogue, #ab17127, ab59286, and Sigma-Aldrich, 2010Catalog, #HPA010694, for example). In some embodiments, ALK-specificantibodies used in the methods of the invention specifically bind thecytoplasmic domain of ALK and, thus, will detect full-length ALK and ALKfusion polypeptides. In some embodiments, ALK-specific antibodies usedin the methods of the invention specifically bind the kinase domain ofALK. Furthermore, ALK fusion-specific antibodies are commerciallyavailable (see CELL SIGNALING TECHNOLOGY, INC., Beverly Mass., 2009/10Catalogue, #s 3343S (phospho-NPM-ALK), 3983 (phospho-NPM-ALK), Abcam,2010 Catalogue, #ab4061 (NPM-ALK), and Thermo Scientific, 2010Catalogue, #PA1-37060 (NPM-ALK), for example). Such antibodies may alsobe produced according to standard methods, as described above.

Detection of expression and/or activity of full-length ALK and/or ALKfusion polypeptide in a biological sample (e.g. a tumor sample) canprovide information on whether the fusion protein alone is driving thetumor, or whether aberrantly expressed full length ALK is also presentand driving the tumor. Such information is clinically useful inassessing whether targeting the fusion protein or the full-lengthprotein(s), or both, or is likely to be most beneficial in inhibitingprogression of the tumor, and in selecting an appropriate therapeutic orcombination thereof. Antibodies specific for the ALK kinaseextracellular domain, which is not present in the mutant ALK disclosedherein, may be particularly useful for determining the presence/absenceof the mutant ALK kinase.

It will be understood that more than one antibody may be used in thepractice of the above-described methods. For example, one or morepolypeptide with ALK kinase activity-specific antibodies together withone or more antibodies specific for full-length ALK kinase, anotherkinase, receptor, or kinase substrate that is suspected of being, orpotentially is, activated in a cancer in which a polypeptide with ALKkinase activity is expressed and/or active may be simultaneouslyemployed to detect the activity of such other signaling molecules in abiological sample comprising cells from such cancer.

Those of skill in the art will appreciate that fusion polypeptides ofthe present invention and the epitope-bearing fragments thereofdescribed above can be combined with parts of other molecules to createchimeric polypeptides. For example, an epitope-bearing fragment of anALK fusion polypeptide may be combined with the constant domain ofimmunoglobulins (IgG) to facilitate purification of the chimericpolypeptide and increase the in vivo half-life of the chimericpolypeptide (see, e.g., examples of CD4-Ig chimeric proteins in EPA394,827; Traunecker et al., Nature 331: 84-86 (1988)). Fusion proteinsthat have a disulfide-linked dimeric structure (e.g., from an IgGportion may also be more efficient in binding and neutralizing othermolecules than the monomeric ALK fusion polypeptide alone (seeFountoulakis et al., J Biochem 270: 3958-3964(1995)).

In some embodiments, the detection molecule used in the methods of theinvention is attached to a detectable label. By “detectable label” withrespect to a polypeptide, polynucleotide, or reagent disclosed hereinmeans a chemical, biological, or other modification of or to thepolypeptide, polynucleotide, or binding agent, including but not limitedto fluorescence, mass, residue, dye, radioisotope, label, or tagmodifications, etc., by which the presence of the molecule of interest(e.g., a polypeptide with ALK kinase activity or a polynucleotideencoding a polypeptide with ALK kinase activity) may be detected. Thedetectable label may be directly or indirectly attached to the detectionmolecule by a covalent or non-covalent chemical bond. Methods forattaching detectable labels to molecules (e.g., to the detectionmolecules described herein) are well known.

Immunoassays useful in the practice of the methods of the invention maybe homogenous immunoassays or heterogeneous immunoassays. In ahomogeneous assay the immunological reaction usually involves a specificreagent (e.g., an ALK-specific antibody), a detectably labeled analyte,and the biological sample of interest. The signal arising from thedetectable label is modified, directly or indirectly, upon the bindingof the antibody to the detectably labeled analyte. Both theimmunological reaction and detection of the extent thereof are carriedout in a homogeneous solution. Immunochemical detectable labels that maybe employed include free radicals, radio-isotopes, fluorescent dyes,enzymes, bacteriophages, coenzymes, and so forth. Semi-conductornanocrystal labels, or “quantum dots”, may also be advantageouslyemployed, and their preparation and use has been well described. Seegenerally, K. Barovsky, Nanotech. Law & Bus. 1(2): Article 14 (2004) andpatents cited therein.

In a heterogeneous assay approach, the reagents are usually thebiological sample, binding reagent (e.g., an antibody), and suitablemeans for producing a detectable signal. Biological samples as furtherdescribed below may be used. The antibody is generally immobilized on asupport, such as a bead, plate or slide, and contacted with the samplesuspected of containing the antigen in a liquid phase. The support isthen separated from the liquid phase and either the support phase or theliquid phase is examined for a detectable signal employing means forproducing such signal. The signal is related to the presence of theanalyte in the biological sample. Means for producing a detectablesignal include the use of radioactive labels, fluorescent labels, enzymelabels, quantum dots, and so forth. For example, if the antigen to bedetected contains a second binding site, an antibody which binds to thatsite can be conjugated to a detectable group and added to the liquidphase reaction solution before the separation step. The presence of thedetectable group on the solid support indicates the presence of theantigen in the test sample. Examples of suitable immunoassays are theradioimmunoassay, immunofluorescence methods, enzyme-linkedimmunoassays, and the like.

Immunoassay formats and variations thereof, which may be useful forcarrying out the methods disclosed herein, are well known in the art.See generally E. Maggio, Enzyme-Immunoassay, (1980) (CRC Press, Inc.,Boca Raton, Fla.); see also, e.g., U.S. Pat. No. 4,727,022 (Skold etal., “Methods for Modulating Ligand-Receptor Interactions and theirapplication”); U.S. Pat. No. 4,659,678 (Forrest et al., “Immunoassay ofAntigens”); U.S. Pat. No. 4,376,110 (David et al., “Immunometric AssaysUsing Monoclonal Antibodies”). Conditions suitable for the formation ofreagent-antibody complexes are well known to those of skill in the art.See id. FN-ALK fusion polypeptide-specific monoclonal antibodies may beused in a “two-site” or “sandwich” assay, with a single hybridoma cellline serving as a source for both the labeled monoclonal antibody andthe bound monoclonal antibody. Such assays are described in U.S. Pat.No. 4,376,110. The concentration of detectable reagent should besufficient such that the binding of a protein with ALK kinase activity(e.g., a full-length ALK protein, a truncated ALK, or ALK fusionpolypeptide) is detectable compared to background.

Antibodies useful in the practice of the methods disclosed herein may beconjugated to a solid support suitable for a diagnostic assay (e.g.,beads, plates, slides or wells formed from materials such as latex orpolystyrene) in accordance with known techniques, such as precipitation.Antibodies or other binding reagents binding reagents may likewise beconjugated to detectable groups such as radiolabels (e.g., ³⁵S, ¹²⁵I,¹³¹I), enzyme labels (e.g., horseradish peroxidase, alkalinephosphatase), and fluorescent labels (e.g., fluorescein) in accordancewith known techniques.

Cell-based assays, such flow cytometry (FC), immuno-histochemistry(IHC), immunocytochemistry (ICC), or immunofluorescence (IF) areparticularly desirable in practicing the methods of the invention, sincesuch assay formats are clinically-suitable, allow the detection ofexpression of a protein with ALK kinase activity (e.g., a mutant ALKpolypeptide or an FN1-ALK fusion polypeptide) in vivo, and avoid therisk of artifact changes in activity resulting from manipulating cellsobtained from, e.g. a tumor sample in order to obtain extracts.Accordingly, in some embodiments, the methods of the invention areimplemented in a flow-cytometry (FC), immunocytochemistry (ICC),immuno-histochemistry (IHC), or immunofluorescence (IF) assay format.

Flow cytometry (FC) may be employed to determine the expression ofpolypeptide with ALK kinase activity in a mammalian tumor before,during, and after treatment with a drug targeted at inhibiting ALKkinase activity. For example, tumor cells from a fine needle aspiratemay be analyzed by flow cytometry for expression and/or activation of apolypeptide with ALK kinase activity or polynucleotide encoding the sameas well as for markers identifying cancer cell types, etc., if sodesired. Flow cytometry may be carried out according to standardmethods. See, e.g. Chow et al., Cytometry (Communications in ClinicalCytometry) 46: 72-78 (2001). Briefly and by way of example, thefollowing protocol for cytometric analysis may be employed: fixation ofthe cells with 2% paraformaldehyde for 10 minutes at 37° C. followed bypermeabilization in 90% methanol for 10 minutes on ice. Cells may thenbe stained with the primary antibody (e.g., a full-length ALK-specificor a ALK fusion polypeptide-specific antibody), washed and labeled witha fluorescent-labeled secondary antibody. The cells would then beanalyzed on a flow cytometer (e.g. a Beckman Coulter FC500) according tothe specific protocols of the instrument used. Such an analysis wouldidentify the level of expressed polypeptide with ALK kinase activity inthe tumor. Similar analysis after treatment of the tumor with anALK-inhibiting therapeutic would reveal the responsiveness of apolypeptide with ALK kinase activity-expressing tumor to the targetedinhibitor of ALK kinase.

Immunohistochemical (IHC) staining may be also employed to determine theexpression and/or activation status of polypeptide with ALK kinaseactivity in a mammalian cancer (e.g., kidney cancer) before, during, andafter treatment with a drug targeted at inhibiting ALK kinase activity(i.e., an ALK-inhibiting therapeutic). IHC may be carried out accordingto well-known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL,Chapter 10, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988).Briefly, and by way of example, paraffin-embedded tissue (e.g. tumortissue from a biopsy) is prepared for immunohistochemical staining bydeparaffinizing tissue sections with xylene followed by ethanol;hydrating in water then PBS; unmasking antigen by heating slide insodium citrate buffer; incubating sections in hydrogen peroxide;blocking in blocking solution; incubating slide in primary antibody(e.g., an ALK-specific or ALK fusion polypeptide-specific antibody) andsecondary antibody; and finally detecting using ABC avidin/biotin methodaccording to manufacturer's instructions.

Immunofluorescence (IF) assays may be also employed to determine theexpression and/or activation status of a polypeptide with ALK kinaseactivity in a mammalian cancer before, during, and after treatment witha drug targeted at inhibiting ALK kinase activity. IF may be carried outaccording to well-known techniques. See, e.g., J. M. polak and S. VanNoorden (1997) INTRODUCTION TO IMMUNOCYTOCHEMISTRY, 2nd Ed.; ROYALMICROSCOPY SOCIETY MICROSCOPY HANDBOOK 37,BioScientific/Springer-Verlag. Briefly, and by way of example, patientsamples may be fixed in paraformaldehyde followed by methanol, blockedwith a blocking solution such as horse serum, incubated with a primaryantibody against (i.e., that specifically binds to) a polypeptide withALK kinase activity followed by a secondary antibody labeled with afluorescent dye such as Alexa 488 and analyzed with an epifluorescentmicroscope.

A variety of other protocols, including enzyme-linked immunosorbentassay (ELISA), radio-immunoassay (RIA), and fluorescent-activated cellsorting (FACS), for measuring expression and/or activity of apolypeptide with ALK kinase activity are known in the art and provide abasis for diagnosing the presence of the polypeptide with ALK kinaseactivity (e.g., full-length ALK, truncated ALK, or an ALK fusionpolypeptide such as an NPM-ALK fusion polypeptide). Normal or standardvalues for polypeptide with ALK kinase activity expression areestablished by combining body fluids or cell extracts taken from normalmammalian subjects, preferably human, with an antibody that specificallybinds to the polypeptide with ALK kinase activity under conditionssuitable for complex formation. The amount of standard complex formationmay be quantified by various methods, but preferably by photometricmeans. Quantities of the polypeptide with ALK kinase activity expressedin subject (i.e., a patient) and control samples from biopsied tissuesare compared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease. Ofcourse, since the polypeptide with ALK kinase activity described hereinare discovered in cancerous kidney cells, no biological samples ofnormal kidney tissue are expected to contain these polypeptide with ALKkinase activity or polynucleotides encoding the same.

In some embodiments, the method for detecting the presence and/oractivity of a polypeptide with ALK kinase activity in a biologicalsample from a mammalian kidney cancer or suspected mammalian kidneycancer comprises the steps of: (a) obtaining a biological sample from amammalian kidney cancer or suspected mammalian kidney cancer and (b)contacting the biological sample with a reagent that detects apolynucleotide encoding the polypeptide with ALK kinase activity,wherein detection of said polynucleotide in said biological sampleindicates said polypeptide with ALK kinase activity is present in saidbiological sample.

As used herein, by “polynucleotide” (or “nucleotide sequence” or“nucleic acid molecule”) refers to a polymer of individual nucleotidescovalently joined together (e.g., via a phosphodiester bond). Thus, thedefinition includes, without limitation, DNA, RNA, genomic DNA, intronicDNA, exonic DNA, cDNA, hnRNA, mRNA, oligonucleotides, or syntheticnucleotides, all of which may be single- or double-stranded, and mayrepresent the sense or anti-sense strand. Probes and primers are withinthe definition of polynucleotides of the invention.

As used herein, by “polynucleotide encoding a polypeptide with ALKkinase activity” is meant to include, without limitation, anypolynucleotide encoding a polypeptide with ALK kinase activity, anypolynucleotide encoding a portion of a polypeptide with ALK kinaseactivity (e.g., the ALK kinase domain), and any polynucleotide from agene encoding a polypeptide with ALK kinase activity regardless ofwhether that particular polynucleotide codes for any amino acid residuesin that polypeptide with ALK kinase activity. For example, intronicsequences from an ALK gene (e.g., from an intron separating two exonscoding for portions of the ALK kinase domain) are included in thedefinition of polynucleotide encoding a polypeptide with ALK kinaseactivity.

The nucleotide sequences, including cDNA and mRNA, of polynucleotidesencoding polypeptides with ALK kinase activity have been previouslypublished. Non-limiting examples include the nucleotide and proteinsequences of human ALK (Genbank Accession Codes: U62540, U66559), thenucleotide and protein sequences of mouse ALK cDNAs (D83002), nucleotideand polypeptide sequences for EML4-ALK (U.S. Pat. No. 7,605,131),EML4-ALK (U.S. Pat. No. 7,700,339), EML4-ALK (GenBank AB462412.1),EML4-ALK (GenBank AB462411.1), KIF5B-ALK (GenBank AB462413.1), andTFG-ALK (GenBank AF143407.1). Furthermore, K. Pulford et al., AnaplasticLymphoma Kinase Proteins in Growth Control and Cancer, J. Cell. Physiol.199, 330-358 (2004) discloses other publicly available ALK fusionpolypeptides.

In various embodiments of all of the aspects of the invention, thereagent that detects a polynucleotide encoding the polypeptide with ALKkinase activity is a nucleic acid probe or primer that hybridizes tosaid polynucleotide. In some embodiments, the nucleic acid probe orprimer hybridizes to the polynucleotide encoding the polypeptide withALK kinase activity under stringent conditions.

As used herein, by “probe,” “primer,” or “oligonucleotide” is meant asingle-stranded nucleic acid molecule of defined sequence that canbase-pair to a second DNA or RNA molecule that contains a complementarysequence (the “target”). The target is generally a nucleic acid ALK geneproduct of an ALK fusion gene. The stability of the resulting hybriddepends upon the extent of the base-pairing that occurs. The extent ofbase-pairing is affected by parameters such as the degree ofcomplementarity between the probe and target molecules, and the degreeof stringency of the hybridization conditions. The degree ofhybridization stringency is affected by parameters such as temperature,salt concentration, and the concentration of organic molecules such asformamide, and is determined by methods known to one skilled in the art.

Probes or primers that specifically bind to a polynucleotide encoding apolypeptide having ALK kinase activity (or a portion of such apolynucleotide) specifically bind the polynucleotide by hybridizing tothe polynucleotide. To do this, the probe or primer that specificallybinds (e.g., hybridizes) to the polynucleotide encoding a polypeptidehaving ALK kinase activity (or a fragment of such a polynucleotide)preferably has at least 50%-55% sequence complementarity, morepreferably at least 60%-75% sequence complementarity, even morepreferably at least 80%-90/o sequence complementarity, yet morepreferably at least 91%-99% sequence complementarity, and mostpreferably 100% sequence complementarity to the polynucleotide encodinga polypeptide having ALK kinase activity (or a fragment of such apolynucleotide) to be detected. Probes, primers, and oligonucleotidesmay be detectably-labeled, either radioactively, or non-radioactively,by methods well-known to those skilled in the art. Probes, primers, andoligonucleotides are used for methods involving nucleic acidhybridization, such as: nucleic acid sequencing, reverse transcriptionand/or nucleic acid amplification by the polymerase chain reaction,single stranded conformational polymorphism (SSCP) analysis, restrictionfragment polymorphism (RFLP) analysis, Southern hybridization, Northernhybridization, in situ hybridization, electrophoretic mobility shiftassay (EMSA).

As used herein, by “hybridizes” is meant that a probe, primer, oroligonucleotide recognizes and physically interact (i.e., formsbase-pairs) with a substantially complementary nucleic acid (e.g., anmRNA encoding full-length ALK or an ALK fusion polypeptide of theinvention) under stringent conditions, and does not substantially basepair with other nucleic acids. A nucleic acid probe, primer, oroligonucleotide that hybridizes under stringent conditions to its targetmay be referred to as a target-specific nucleic acid probe (or primer oroligonucleotide) or an anti-target nucleic acid probe (or primer oroligonucleotide). For example, a nucleic acid probe that hybridizes to apolynucleotide encoding an EML4-ALK fusion polypeptide may be referredto as an EML4-ALK-specific nucleic acid probe or an anti-EML4-ALKnucleic acid probe.

As used herein, the term “stringent conditions” with respect tonucleotide sequence or nucleotide probe hybridization conditions is the“stringency” that occurs within a range from about T_(m) minus 5° C.(i.e., 5° C. below the melting temperature (T_(m)) of the reagent ornucleic acid probe) to about 20° C. to 25° C. below T_(m). Typicalstringent conditions are: overnight incubation at 42° C. in a solutioncomprising: 50% formamide, 5 X.SSC (750 mM NaCl, 75 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 micrograms/ml denatured, sheared salmon spermDNA, followed by washing the filters in 0.1×SSC at about 65° C. As willbe understood by those of skill in the art, the stringency ofhybridization may be altered in order to identify or detect identical orrelated polynucleotide sequences. For example, for a DNA probe of atleast 500 nucleotides in length, stringent conditions may be achieved byhybridization occurring in a buffer containing 0.5 M NaHPO₄, pH 7.2, 7%SDS, 1 mM EDTA, and 1% BSA (fraction V), at a temperature of 65° C., ora buffer containing 48% formamide, 4.8×SSC, 0.2 M Tris-Cl, pH 7.6, lxDenhardt's solution, 10% dextran sulfate, and 0.1% SDS, at a temperatureof 42° C. (or similarly typical conditions for stringenct Northern orSouthern hybridizations). Stringent hybridization is relied upon for thesuccess of numerous techniques routinely performed by molecularbiologists, such as high stringency PCR, DNA sequencing, single strandconformational polymorphism analysis, and in situ hybridization. Incontrast to Northern and Southern hybridizations, these techniques areusually performed with relatively short probes (e.g., usually 16nucleotides or longer for PCR or sequencing, and 40 nucleotides orlonger for in situ hybridization). The stringent conditions used inthese techniques are well known to those skilled in the art of molecularbiology, and may be found, for example, in F. Ausubel et al., CurrentProtocols in Molecular Biology, supra, herein incorporated by reference.

In various embodiments of all of the aspects of the invention, thenucleic acid probe or primer is a polymerase chain reaction (PCR) probe,a fluorescence in situ hybridization (FISH) probe, or a Southernblotting probe. In some embodiments, the method is implemented in apolymerase chain reaction (PCR) assay format, a in situ hybridization(ISH) assay format, or a Southern blotting assay format.

Polynucleotides encoding a polypeptide with ALK kinase activity may alsobe used for diagnostic purposes. The polynucleotides that may be usedinclude oligonucleotide sequences, antisense RNA and DNA molecules, andPNAs. The polynucleotides may be used to detect and quantitate geneexpression in biopsied tissues in which expression of a polypeptide withALK kinase activity (e.g., full length ALK, or an ALK fusionpolypeptide, The diagnostic assay may be used to distinguish betweenabsence, presence, and aberrant expression of a polypeptide with ALKkinase activity, and to monitor regulation of levels of a polypeptidewith ALK kinase activity during therapeutic intervention.

In one embodiment, hybridization with PCR probes which are capable ofdetecting a polynucleotide, including genomic sequences, encoding apolypeptide with ALK kinase activity may be used to identifypolynucleotides that encode such polypeptides with ALK kinase activity.The construction and use of such probes is described herein. Thespecificity of the probe, whether it is made from a highly specificregion, e.g., 10 unique nucleotides in the fusion junction, or a lessspecific region, e.g., the 3′ coding region, and the degree of thehybridization or amplification (e.g., stringent hybridization or notstringent) will determine whether the probe identifies only naturallyoccurring sequences encoding mutant ALK kinase polypeptide, alleles, orrelated sequences. In some embodiments, nucleic acid probes useful inthe methods described herein may hybridize to nucleotide sequencesencoding the kinase domain of ALK (amino acids 1116-1392 of SEQ IDNO:2). The probes may alternatively hybridize nucleotides encoding theC-terminal domain located at amino acids 1376-1620 of SEQ ID NO: 2,amino acid residues 1504-1507 of SEQ ID NO:2 making up thephosphotyrosine-binding site of the C-terminal domain of ALK, or aminoacid residues 1603-1606 of SEQ ID NO: 2 representing the interactionsite for the phosphotyrosine-dependent binding of the substratephosphlipase C-γ (PLC-γ).

In another embodiment of the invention, the polynucleotides encoding apolypeptide with ALK kinase activity may be used to generatehybridization probes which are useful for mapping the naturallyoccurring genomic sequence. The sequences may be mapped to a particularchromosome or to a specific region of the chromosome using well knowntechniques. Such techniques include in-situ hybridization (ISH), FACS,or artificial chromosome constructions, such as yeast artificialchromosomes, bacterial artificial chromosomes, bacterial P1constructions or single chromosome cDNA libraries, as reviewed in Price,C. M., Blood Rev. 7: 127-134 (1993), and Trask, B. J., Trends Genet. 7:149-154 (1991).

In situ hybridization (ISH) of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, AT to 11q22-23 (Gatti et al.,Nature 336: 577-580 (1988)), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc., among normal, carrier, or affected individuals.

In one embodiment, fluorescence in-situ hybridization (FISH) (anon-limiting type of in situ hybridization assay) is employed (asdescribed in Verma et al. HUMAN CHROMOSOMES: A MANUAL OF BASICTECHNIQUES, Pergamon Press, New York, N.Y. (1988)) and may be correlatedwith other physical chromosome mapping techniques and genetic map data.The FISH technique is well known (see, e.g., U.S. Pat. Nos. 5,756,696;5,447,841; 5,776,688; and 5,663,319). Examples of genetic map data canbe found in the 1994 Genome Issue of Science (265: 1981f). Correlationbetween the location of the gene encoding ALK protein and/or the geneencoding the fusion partner of an ALK fusion polypeptide on a physicalchromosomal map and a specific disease, or predisposition to a specificdisease, may help delimit the region of DNA associated with that geneticdisease. The nucleotide sequences of the subject invention may be usedto detect differences in gene sequences between normal, carrier, oraffected individuals. FISH protocols for detect translocations involvingthe ALK gene have been described (see, e.g., U.S. Pat. No. 7,700,339 andUS Patent Publication No. 20110110923. A dual color, break apart probedesigned to detect ALK gene translocation is also commercially availablefrom Abbott Molecular (Abbott Park, Ill., US; Catalog No. 05J89-001).

It shall be understood that all of the methods (e.g., PCR and FISH) thatdetect a polynucleotide encoding a polypeptide with ALK kinase activitymay be combined with other methods that detect expression and/oractivity of a polypeptide with ALK kinase activity. For example,detection of a polynucleotide encoding an ALK fusion polypeptide in thegenetic material of a biological sample (e.g., TFG-ALK in a circulatingtumor cell) may be followed by Western blotting analysis orimmuno-histochemistry (IHC) analysis of the proteins of the sample todetermine if the polynucleotide encoding the TFG-ALK fusion polypeptideis actually expressed as a TFG-ALK fusion polypeptide in the biologicalsample. Such Western blotting or IHC analyses may be performed using anantibody that specifically binds to the polypeptide encoded by thedetected polynucleotide, or the analyses may be performed usingantibodies that specifically bind either to full length TFG (e.g., bindto the N-terminus of the TFG protein) or to full length ALK (e.g., bindan epitope in the kinase domain of ALK protein). Such assays are knownin the art (see, e.g., U.S. Pat. No. 7,468,252).

In another example, the CISH technology of Dako allows chromatogenic insitu hybridization with immuno-histochemistry on the same tissuesection. See Elliot et al., Br J Biomed Sci 2008; 65(4): 167-171, 2008for a comparison of CISH and FISH.

In another aspect, the invention relates to detecting the detectionmolecules utilizing a detection device. By “detection device” is meantany device that is used to detect, measure, or otherwise quantify theexpression and/or activity levels of the polypeptide with ALK kinaseactivity or polynucleotide encoding the polypeptide with ALK kinaseactivity. The detected levels of expression and/or activity may be usedto generate a database of the samples, particularly of the test andcontrol samples. The database can be used to generate a report toanalyze and compare the levels of expression and/or activity of thepolypeptide with ALK kinase activity or polynucleotide encoding thepolypeptide with ALK kinase activity of the samples. Non-limitingexamples of detection devices include a PCR cycler, DNA analyzer, DNAsequencer, DNA extraction column, gel, or kit, image analysis device,chromatographic device, or mass spectrometer, combinations thereof, andautomated versions thereof. An image analysis device is any device thatis used to create a visible image of the biological sample for detectionof the polypeptide with ALK kinase activity or polynucleotide encodingthe polypeptide with ALK kinase activity and includes the followingnon-limiting examples: FlowCAM, a biosensor imaging device, an infraredimaging systems, or a chemiluminescent imaging systems. Achromatographic device may alternatively be used to detect the ALK geneproduct, which can perform high performance liquid chromatography(HPLC), reverse HPLC, gas chromatography, or liquid chromatography. Inanother embodiment, a mass spectrometer may be utilized.

In one embodiment, polypeptide with ALK kinase activity orpolynucleotide encoding the polypeptide with ALK kinase activity aredetected in a biological sample using a mass spectrometer. The term“mass spectrometer” (MS) means a device capable of detecting specificmolecular species and measuring their accurate masses. The term is meantto include any molecular detector into which a polypeptide or peptidemay be eluted for detection and/or characterization and includes, forexample, MALDI and SELDI devices. In the preferred MS procedure, asample, e.g., the elution solution, is loaded onto the MS instrument,and undergoes vaporization. The components of the sample are ionized byone of a variety of methods (e.g., by electrospray ionization or “ESI”),which results in the formation of positively charged particles (ions).The positive ions are then accelerated by a magnetic field. Thecomputation of the mass-to-charge ratio of the particles is based on thedetails of motion of the ions as they transit through electromagneticfields, and detection of the ions. The preferred mass measurement errorof a mass spectrometer of the invention is 10 ppm or less, morepreferable is 7 ppm or less; and most preferably 5 ppm or less.

Fragment ions in the MS/MS and MS³ spectra are generally highly specificand diagnostic for peptides of interest. In contrast, to prior methods,the identification of peptide diagnostic signatures provides for a wayto perform highly selective analysis of a complex protein mixture, suchas a cellular lysate in which there may be greater than about 100, about1000, about 10,000, or even about 100,000 different kinds of proteins.Thus, while conventional mass spectroscopy would not be able todistinguish between peptides with different sequences but similar m/zratios (which would tend to co-elute with any labeled standard beinganalyzed), the use of peptide fragmentation methods and multistage massspectrometry in conjunction with LC methods, provide a way to detect andquantify target proteins which are only a small fraction of a complexmixture (e.g., present in less than 2000 copies per cell or less thanabout 0.001% of total cellular protein) through these diagnosticsignatures.

Test peptides in a biological sample are preferably examined bymonitoring of a selected reaction in the mass spectrometer. Thisinvolves using the prior knowledge gained by the characterization of astandard peptide and then requiring the mass spectrometer tocontinuously monitor a specific ion in the MS/MS or MS' spectrum forboth the peptide of interest and the standard peptide. After elution,the areas-under-the-curve (AUC) for both the standard peptide and targetpeptide peaks may be calculated. The ratio of the two areas provides theabsolute quantification that may then be normalized for the number ofcells used in the analysis and the protein's molecular weight, toprovide the precise number of copies of the protein per cell.

As used herein the term, “accurate mass” refers to an experimentally ortheoretically determined mass of an ion that is used to determine anelemental formula. For ions containing combinations of the elements C,H, N, O, P, S, and the halogens, with mass less than 200 Unified AtomicMass Units, a measurement about 5 ppm uncertainty is sufficient touniquely determine the elemental composition.

As used herein the term, “predetermined peptide accurate mass” refers tothe experimentally determined or calculated accurate mass of a peptidewith a known amino acid sequence (along with any associatedpost-translational modifications). The accurate mass of any suchspecific amino acid sequence may be readily calculated by one of skillin the art.

As used herein, “a peptide fragmentation signature” refers to thedistribution of mass-to-charge ratios of fragmented peptide ionsobtained from fragmenting a peptide, for example, by collision induceddisassociation, ECD, LID, PSD, IRNPD, SID, and other fragmentationmethods. A peptide fragmentation signature which is “diagnostic” or a“diagnostic signature” of a target protein or target polypeptide is onewhich is reproducibly observed when a peptide digestion product of atarget protein/polypeptide identical in sequence to the peptide portionof a standard peptide, is fragmented and which differs only from thefragmentation pattern of the standard peptide by the mass of themass-altering label and/or the presence of a ubiquitin remnant.Preferably, a diagnostic signature is unique to the target protein(i.e., the specificity of the assay is at least about 95%, at leastabout 99%, and preferably, approaches 100%).

In some embodiments, the mammalian kidney cancer or suspected mammaliankidney cancer in which the presence or activity of said polypeptide withALK kinase activity is detected is identified as a mammalian kidneycancer or suspected mammalian kidney cancer likely to respond to anALK-inhibiting therapeutic.

As used herein, by “likely to respond” is meant that a cancer is morelikely to show growth retardation or growth abrogation in response to(e.g., upon contact with or treatment by) an ALK inhibitor (alsoreferred to as an ALK-inhibiting therapeutic) as compared to anuntreated cancer (e.g., of the same tissue type as the treated cancer).In some embodiments, a cancer that is likely to respond to an ALKinhibitor is one that shrinks in size (e.g., the cancer cells apoptose)in response to the ALK inhibitor as compared to an untreated cancer. Insome embodiments, a cancer that is likely to respond to an ALK inhibitoris one that dies (e.g., the cancer cells apoptose) in response to theALK inhibitor as compared to an untreated cancer.

Accordingly, should a patient or subject whose kidney cancer orsuspected kidney cancer is identified as comprising a polypeptide withALK kinase activity (e.g., by detection of ALK kinase activity, apolypeptide with ALK kinase activity, and/or a polynucleotide encoding apolypeptide with ALK kinase activity), that patient may be treated with(e.g., administered with) a therapeutically effective amount of anALK-inhibiting therapeutic. In some embodiments, the ALK-inhibitingtherapeutic is administered in a pharmaceutically acceptableformulation.

As used herein, by “therapeutically effective amount” or“pharmaceutically effective amount” is mean an amount of anALK-inhibiting therapeutic that is adequate to inhibit the cancer (orcell thereof) or suspected cancer (or cells thereof), as compared to anuntreated cancer or suspected cancer, by either slowing the growth ofthe cancer or suspected cancer, reducing the mass of the cancer orsuspected cancer, reducing the number of cells of the cancer orsuspected cancer, or killing the cancer.

An ALK-inhibiting therapeutic may be any composition comprising at leastone ALK inhibitor. Such compositions also include compositionscomprising only a single ALK-inhibiting compound, as well ascompositions comprising multiple therapeutics (including those againstother RTKs), which may also include a non-specific therapeutic agentlike a chemotherapeutic agent or general transcription inhibitor.

In some embodiments, an ALK-inhibiting therapeutic useful in thepractice of the methods of the invention is a targeted, small moleculeinhibitor. Small molecule targeted inhibitors are a class of moleculesthat typically inhibit the activity of their target enzyme byspecifically, and often irreversibly, binding to the catalytic site ofthe enzyme, and/or binding to an ATP-binding cleft or other binding sitewithin the enzyme that prevents the enzyme from adopting a conformationnecessary for its activity.

Crizotinib (also known as PF-02341066 or 1066), is a c-MET/HGFR and ALK(anaplastic lymphoma kinase) inhibitor of the aminopyridine chemicalseries that is being developed by Pfizer Incorporated (see Zou et al.,Cancer Research 67: 4408-4417, 2007 and supplemental data). Crizotinibis currently undergoing clinical trials testing its safety and efficacyin treating several forms of cancer, particularly non-small cell lungcarcinoma (NSCLC), anaplastic large cell lymphoma, neuroblastoma, andother advanced solid tumors in both adults and children.

U.S. Pub. No. 2008/0300273 discloses that PF-02341066 was able to reducecell colony scattering in HGF-stimulated Madin-Darby Canine Kidney(MDCK) cells. As such, the compound was able to inhibit epithelial celldispersion and motility in response to HGF. See also Zou et al., CancerResearch 67: 4408-4417, 2007. However, these publications do notdisclose whether or not ALK or ALK fusions are actually expressed inkidney cells or kidney cancer cells. In fact, Zou et al., CancerResearch 67: 4408-4417, 2007 references Christensen et al., Cancer Res.63: 7345, 2003 as describing a cMET-inhibiting assay using the MDCK cellmodel. To determine whether or not HGF-stimulated MDCK cells actuallyexpress ALK kinase, the present inventors replicated the conditionsdescribed in U.S. Pub. No. 2008/0300273. As described below in Example3, using Western blotting analysis, it was determined thatHGF-stimulated MDCK cells do not express any ALK kinase. The cells didnot express phosphorylated or non-phosphorylated ALK kinase.Accordingly, the effects on HGF-stimulated MDCK cells in response tocrizotinib described in U.S. Pub. No. 2008/0300273 could not have beenmediated by the ALK kinase since ALK kinase was not present in thecells. Accordingly, as proposed in U.S. Pub. No. 2008/0300273, theeffects of crizotinib on HGF-stimulated MDCK cells were mediated throughMET kinase.

Additional small molecule kinase inhibitors that may target ALK includeTAE-684 (from Novartis; see Galkin, et al., Proc. National Acad. Sci104(1) 270-275, 2007), AP26113 (Ariad Pharmaceuticals, Inc.), andCEP-14083, CEP-14513, and CEP-11988 (Cephalon; see Wan et al., Blood107: 1617-1623, 2006); and WHI-P131 and WHI-P154 (EMD Biosciences; seeMarzec et al., (Laboratory Investigation: A journal of Technical Methodsand Pathology 2005, Vol. 85, p. 1544-1554, 2005). Another group hasdeveloped their own low-molecular-weight ALK-inhibiting substance andhas demonstrated that this inhibitor induces the cell death ofNPM-ALK-expressing lymphoma cell lines (Blood, 2006, Vol. 107, p.1617-1623). In addition, numerous other compounds having an inhibitoryactivity against ALK have been reported including5-chloro-N⁴-[2-(isopropylsulfonyl)phenyl]-N₂-{2-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidine-2,4-diamineand2-[(5-bromo-2-{[2-methoxy-4-(4-methylpiperazin-1-yl)phenyl]amino}pyrimidi-n-4-yl)amino]-N-methylbenzenesulfonamide(see Mosse et al., Clin Cancer Res. 2009 Sep. 15; 15(18):5609-14, 2009;Journal of Medicinal Chemistry 49: 1006-1015, 2006; Cancer Research,(US), 2004, Vol. 64, p. 8919-8923, 2004; Proc. Natl Acad. Sci.101:13306-13311, 2004; Annual Review of Medicine, (US) 54: 73, 2003;Laboratory Investigation; A Journal of Technical Methods and Pathology,(US) 83: 1255-1265, 2003; Cellular and Molecular Life Sciences 61:2897-2911, 2004; Science 278: 1309-1312, 1997; Oncogene 14 (4): 439-449,1997; Oncogene 9: 1567-1574, 1994; Am J Pathol 160: 1487-1494, 2002; AmJ Pathol 157: 377-384, 2000; Blood 90: 2901-2910, 1997; Am J Pathol. 156(3): 781-9, 2000; J Comb Chem. 8: 401-409, 2006 and U.S. Pub. Nos.20100152182; 20100099658; 20100048576; 20090286778; 20090221555;20090186801; 20090118216; 20090099193; 20080176881; 20080090776;2008/0300273; WO 2005/097765; WO 2005/009389; WO 2005/016894; WO2004/080980; and WO2004079326.

Additional small molecule inhibitors and other inhibitors (e.g.,indirect inhibitors) of ALK kinase activity may be rationally designedusing X-ray crystallographic or computer modeling of ALK threedimensional structure, or may found by high throughput screening ofcompound libraries for inhibition of key upstream regulatory enzymesand/or necessary binding molecules, which results in inhibition of ALKkinase activity. Such approaches are well known in the art, and havebeen described. ALK inhibition by such therapeutics may be confirmed,for example, by examining the ability of the compound to inhibit ALKactivity, but not other kinase activity, in a panel of kinases, and/orby examining the inhibition of ALK activity in a biological samplecomprising cancer cells (e.g., kidney cancer cells). Methods foridentifying compounds that inhibit a cancer characterized by theexpression/presence of polypeptide with ALK kinase activity, are furtherdescribed below.

ALK-inhibiting therapeutics useful in the methods of the invention mayalso be targeted antibodies that specifically bind to critical catalyticor binding sites or domains required for ALK activity, and inhibit thekinase by blocking access of ligands, substrates or secondary moleculesto a and/or preventing the enzyme from adopting a conformation necessaryfor its activity. The production, screening, and therapeutic use ofhumanized target-specific antibodies has been well-described. SeeMerluzzi et al., Adv Clin Path. 4(2): 77-85 (2000). Commercialtechnologies and systems, such as Morphosys, Inc.'s Human CombinatorialAntibody Library (HuCAL®), for the high-throughput generation andscreening of humanized target-specific inhibiting antibodies areavailable.

The production of various anti-receptor kinase targeted antibodies andtheir use to inhibit activity of the targeted receptor has beendescribed. See, e.g. U.S. Patent Publication No. 20040202655, U.S.Patent Publication No. 20040086503, U.S. Patent Publication No.20040033543, Standardized methods for producing, and using, receptortyrosine kinase activity-inhibiting antibodies are known in the art.See, e.g., European Patent No. EP1423428,

Phage display approaches may also be employed to generate ALK-specificantibody inhibitors, and protocols for bacteriophage libraryconstruction and selection of recombinant antibodies are provided in thewell-known reference text CURRENT PROTOCOLS IN IMMUNOLOGY, Colligan etal. (Eds.), John Wiley & Sons, Inc. (1992-2000), Chapter 17, Section17.1. See also U.S. Pat. Nos. 6,319,690, 6,300,064, 5,840,479, and U.S.Patent Publication No. 20030219839.

ALK-binding targeted antibodies identified in screening of antibodylibraries as describe above may then be further screened for theirability to block the activity of ALK, both in vitro kinase assay and invivo in cell lines and/or tumors. ALK inhibition may be confirmed, forexample, by examining the ability of such antibody therapeutic toinhibit ALK kinase activity in a panel of kinases, and/or by examiningthe inhibition of ALK activity in a biological sample comprising cancercells, as described above. In some embodiments, a ALK-inhibitingcompound of the invention reduces ALK kinase activity, but reduces thekinase activity of other kinases to a lesser extent (or not at all).Methods for screening such compounds for ALK kinase inhibition arefurther described above.

ALK-inhibiting compounds that useful in the practice of the disclosedmethods may also be compounds that indirectly inhibit ALK activity byinhibiting the activity of proteins or molecules other than ALK kinaseitself. Such inhibiting therapeutics may be targeted inhibitors thatmodulate the activity of key regulatory kinases that phosphorylate orde-phosphorylate (and hence activate or deactivate) ALK itself, orinterfere with binding of ligands. As with other receptor tyrosinekinases, ALK regulates downstream signaling through a network of adaptorproteins and downstream kinases. As a result, induction of cell growthand survival by ALK activity may be inhibited by targeting theseinteracting or downstream proteins.

ALK kinase activity may also be indirectly inhibited by using a compoundthat inhibits the binding of an activating molecule necessary for fulllength ALK, an ALK fusion polypeptide (e.g., an FN1-ALK fusionpolypeptide), or mutant ALK (e.g., a truncated ALK polypeptide or anFN1-tmALK fusion polypeptide) to adopt its active conformation. Forexample, the production and use of anti-PDGF antibodies has beendescribed. See U.S. Patent Publication No. 20030219839, “Anti-PDGFAntibodies and Methods for Producing Engineered Antibodies,” Bowdish etal. Inhibition of ligand (PDGF) binding to the receptor directlydown-regulates the receptor activity.

ALK inhibiting compounds or therapeutics may also comprise anti-senseand/or transcription inhibiting compounds that inhibit ALK kinaseactivity by blocking transcription of the gene encoding ALK, an FN1-ALKfusion-encoding gene, or a mutant ALK-encoding gene. The inhibition ofvarious receptor kinases, including VEGFR, EGFR, and IGFR, and FGFR, byantisense therapeutics for the treatment of cancer has been described.See, e.g., U.S. Pat. Nos. 6,734,017; 6, 710,174, 6,617,162; 6,340,674;5,783,683; 5,610,288.

Antisense oligonucleotides may be designed, constructed, and employed astherapeutic agents against target genes in accordance with knowntechniques. See, e.g. Cohen, J., Trends in Pharmacol. Sci. 10(11):435-437 (1989); Marcus-Sekura, Anal. Biochem. 172: 289-295 (1988);Weintraub, H., Sci. AM. pp. 40-46 (1990); Van Der Krol et al.,BioTechniques 6(10): 958-976 (1988); Skorski et al., Proc. Nat. Acad.Sci. US (1994) 91: 4504-4508. Inhibition of human carcinoma growth invivo using an antisense RNA inhibitor of EGFR has recently beendescribed. See U.S. Patent Publication No. 20040047847. Similarly, aALK-inhibiting therapeutic comprising at least one antisenseoligonucleotide against a mammalian ALK gene, FN1-ALK fusionpolynucleotide or mutant ALK polynucleotide may be prepared according tomethods described above. Pharmaceutical compositions comprisingALK-inhibiting antisense compounds may be prepared and administered asfurther described below.

Small interfering RNA molecule (siRNA) compositions, which inhibittranslation, and hence activity, of ALK through the process of RNAinterference, may also be desirably employed in the methods of theinvention. RNA interference, and the selective silencing of targetprotein expression by introduction of exogenous small double-strandedRNA molecules comprising sequence complimentary to mRNA encoding thetarget protein, has been well described. See, e.g. U.S. PatentPublication No. 20040038921, U.S. Patent Publication No. 20020086356,and U.S. Patent Publication 20040229266.

Double-stranded RNA molecules (dsRNA) have been shown to block geneexpression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). Briefly, the RNAse III Dicer processes dsRNA intosmall interfering RNAs (siRNA) of approximately 22 nucleotides, whichserve as guide sequences to induce target-specific mRNA cleavage by anRNA-induced silencing complex RISC (see Hammond et al., Nature (2000)404: 293-296). RNAi involves a catalytic-type reaction whereby newsiRNAs are generated through successive cleavage of longer dsRNA. Thus,unlike antisense, RNAi degrades target RNA in a non-stoichiometricmanner. When administered to a cell or organism, exogenous dsRNA hasbeen shown to direct the sequence-specific degradation of endogenousmessenger RNA (mRNA) through RNAi.

A wide variety of target-specific siRNA products, including vectors andsystems for their expression and use in mammalian cells, are nowcommercially available (e.g., Promega, Inc.; and Dharmacon, Inc.Detailed technical manuals on the design, construction, and use of dsRNAfor RNAi are available. See, e.g., Dharmacon's “RNAi Technical Reference& Application Guide”; Promega's “RNAi: A Guide to Gene Silencing.”ALK-inhibiting siRNA products are also commercially available, and maybe suitably employed in the method of the invention. See, e.g.,Dharmacon, Inc., Lafayette, Colo. (Cat Nos. M-003162-03, MU-003162-03,D-003162-07 thru −10 (siGENOME™ SMARTselection and SMARTpool● siRNAs).

It has recently been established that small dsRNA less than 49nucleotides in length, and preferably 19-25 nucleotides, comprising atleast one sequence that is substantially identical to part of a targetmRNA sequence, and which dsRNA optimally has at least one overhang of1-4 nucleotides at an end, are most effective in mediating RNAi inmammals. See U.S. Patent Publication Nos. 20040038921 and 20040229266.The construction of such dsRNA, and their use in pharmaceuticalpreparations to silence expression of a target protein, in vivo, aredescribed in detail in such publications.

If the sequence of the gene to be targeted in a mammal is known, 21-23nt RNAs, for example, can be produced and tested for their ability tomediate RNAi in a mammalian cell, such as a human or other primate cell.Those 21-23 nt RNA molecules shown to mediate RNAi can be tested, ifdesired, in an appropriate animal model to further assess their in vivoeffectiveness. Target sites that are known, for example target sitesdetermined to be effective target sites based on studies with othernucleic acid molecules, for example ribozymes or antisense, or thosetargets known to be associated with a disease or condition such as thosesites containing mutations or deletions, can be used to design siRNAmolecules targeting those sites as well.

Alternatively, the sequences of effective dsRNA can be rationallydesigned/predicted screening the target mRNA of interest for targetsites, for example by using a computer folding algorithm. The targetsequence can be parsed in silico into a list of all fragments orsubsequences of a particular length, for example 23 nucleotidefragments, using a custom Perl script or commercial sequence analysisprograms such as Oligo, MacVector, or the GCG Wisconsin Package.

Various parameters can be used to determine which sites are the mostsuitable target sites within the target RNA sequence. These parametersinclude but are not limited to secondary or tertiary RNA structure, thenucleotide base composition of the target sequence, the degree ofhomology between various regions of the target sequence, or the relativeposition of the target sequence within the RNA transcript. Based onthese determinations, any number of target sites within the RNAtranscript can be chosen to screen siRNA molecules for efficacy, forexample by using in vitro RNA cleavage assays, cell culture, or animalmodels. See, e.g., U.S. Patent Publication No. 20030170891. An algorithmfor identifying and selecting RNAi target sites has also recently beendescribed. See U.S. Patent Publication No. 20040236517.

Commonly used gene transfer techniques include calcium phosphate,DEAE-dextran, electroporation and microinjection and viral methods(Graham et al. (1973) Virol. 52: 456; McCutchan et al., (1968), J. Natl.Cancer Inst. 41: 351; Chu et al. (1987), Nucl. Acids Res. 15: 1311;Fraley et al. (1980), J. Biol. Chem. 255: 10431; Capecchi (1980), Cell22: 479). DNA may also be introduced into cells using cationic liposomes(Feigner et al. (1987), Proc. Natl. Acad. Sci LISA 84: 7413).Commercially available cationic lipid formulations include Tfx 50(Promega) or Lipofectamin 200 (Life Technologies). Alternatively, viralvectors may be employed to deliver dsRNA to a cell and mediate RNAi. SeeU.S. Patent Publication No. 20040023390.

Transfection and vector/expression systems for RNAi in mammalian cellsare commercially available and have been well described. See, e.g.,Dharmacon, Inc., DharmaFECT™ system; Promega, Inc., siSTRIKE™ U6 Hairpinsystem; see also Gou et al. (2003) FEBS. 548, 113-118; Sui, G. et al. ADNA vector-based RNAi technology to suppress gene expression inmammalian cells (2002) Proc. Natl. Acad. Sci. 99, 5515-5520; Yu et al.(2002) Proc. Natl. Acad. Sci. 99, 6047-6052; Paul, C. et al. (2002)Nature Biotechnology 19, 505-508; McManus et al. (2002) RNA 8, 842-850.

siRNA interference in a mammal using prepared dsRNA molecules may thenbe effected by administering a pharmaceutical preparation comprising thedsRNA to the mammal. The pharmaceutical composition is administered in adosage sufficient to inhibit expression of the target gene. dsRNA cantypically be administered at a dosage of less than 5 mg dsRNA perkilogram body weight per day, and is sufficient to inhibit or completelysuppress expression of the target gene. In general a suitable dose ofdsRNA will be in the range of 0.01 to 2.5 milligrams per kilogram bodyweight of the recipient per day, preferably in the range of 0.1 to 200micrograms per kilogram body weight per day, more preferably in therange of 0.1 to 100 micrograms per kilogram body weight per day, evenmore preferably in the range of 1.0 to 50 micrograms per kilogram bodyweight per day, and most preferably in the range of 1.0 to 25 microgramsper kilogram body weight per day. A pharmaceutical compositioncomprising the dsRNA is administered once daily, or in multiplesub-doses, for example, using sustained release formulations well knownin the art. The preparation and administration of such pharmaceuticalcompositions may be carried out accordingly to standard techniques, asfurther described below.

Such dsRNA may then be used to inhibit ALK expression and activity in acancer, by preparing a pharmaceutical preparation comprising atherapeutically-effective amount of such dsRNA, as described above, andadministering the preparation to a human subject having a cancer (e.g.,kidney cancer) expressing an ALK fusion protein or aberrantly expressingfull length ALK polypeptide, for example, via direct injection to thetumor. The similar inhibition of other receptor tyrosine kinases, suchas VEGFR and EGFR using siRNA inhibitors has been described. See U.S.Patent Publication No. 20040209832, U.S. Patent Publication No.20030170891, and U.S. Patent Publication No. 20040175703.

ALK inhibitors (or their pharmaceutically acceptable salts) are to beprovided to patients in an amount/dosage, frequency and form (includingstandard excipients if needed) as determined by a competent clinician orpharmacist. The inhibitors may be provided orally, parentally,intravenously, for example. If the PF02341066 is used as an ALKinhibitor in the context of the invention, guidance for its delivery,dosage and formulation may be found in U.S. Pub. No. 2008/0300273, forexample.

Of course ALK-inhibiting therapeutic compositions useful in the practiceof the methods of the invention may be administered to a mammal by anymeans known in the art including, but not limited to oral or peritonearoutes, including intravenous, intramuscular, intraperitoneal,subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical(including buccal and sublingual) administration.

For oral administration, a ALK-inhibiting therapeutic will generally beprovided in the form of tablets or capsules, as a powder or granules, oras an aqueous solution or suspension. Tablets for oral use may includethe active ingredients mixed with pharmaceutically acceptable carriersand excipients such as inert diluents, disintegrating agents, bindingagents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract.

Capsules for oral use include hard gelatin capsules in which the activeingredient is mixed with a solid diluent, and soft gelatin capsuleswherein the active ingredients is mixed with water or an oil such aspeanut oil, liquid paraffin or olive oil. For intramuscular,intraperitoneal, subcutaneous and intravenous use, the pharmaceuticalcompositions of the invention will generally be provided in sterileaqueous solutions or suspensions, buffered to an appropriate pH andisotonicity. Suitable aqueous vehicles include Ringer's solution andisotonic sodium chloride. The carrier may consist exclusively of anaqueous buffer (“exclusively” means no auxiliary agents or encapsulatingsubstances are present which might affect or mediate uptake of theALK-inhibiting therapeutic). Such substances include, for example,micellar structures, such as liposomes or capsids, as described below.Aqueous suspensions may include suspending agents such as cellulosederivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth,and a wetting agent such as lecithin. Suitable preservatives for aqueoussuspensions include ethyl and n-propyl p-hydroxybenzoate.

ALK-inhibiting therapeutic compositions may also include encapsulatedformulations to protect the therapeutic (e.g., a dsRNA compound or anantibody that specifically binds an ALK fusion polypeptide) againstrapid elimination from the body, such as a controlled releaseformulation, including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811; PCT publication WO91/06309; and European patent publication EP-A-43075. An encapsulatedformulation may comprise a viral coat protein. The viral coat proteinmay be derived from or associated with a virus, such as a polyoma virus,or it may be partially or entirely artificial. For example, the coatprotein may be a Virus Protein 1 and/or Virus Protein 2 of the polyomavirus, or a derivative thereof.

ALK-inhibiting compounds can also comprise a delivery vehicle, includingliposomes, for administration to a subject, carriers and diluents andtheir salts, and/or can be present in pharmaceutically acceptableformulations. For example, methods for the delivery of nucleic acidmolecules are described in Akhtar et al., 1992, Trends Cell Bio., 2,139; DELIVERY STRATEGIES FOR ANTISENSE OLIGONUCLEOTIDE THERAPEUTICS, ed.Akbtar, 1995, Maurer et al., 1999, Mol. Membr. Biol., 16, 129-140;Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137, 165-192; and Leeet al., 2000, ACS Symp. Ser., 752, 184-192. U.S. Pat. No. 6,395,713 andPCT Publication No. WO 94/02595 further describe the general methods fordelivery of nucleic acid molecules. These protocols can be utilized forthe delivery of virtually any nucleic acid molecule.

ALK-inhibiting therapeutics can be administered to a mammalian tumor bya variety of methods known to those of skill in the art, including, butnot restricted to, encapsulation in liposomes, by iontophoresis, or byincorporation into other vehicles, such as hydrogels, cyclodextrins,biodegradable nanocapsules, and bioadhesive microspheres, or byproteinaceous vectors (see PCT Publication No. WO 00/53722).Alternatively, the therapeutic/vehicle combination is locally deliveredby direct injection or by use of an infusion pump. Direct injection ofthe composition, whether subcutaneous, intramuscular, or intradermal,can take place using standard needle and syringe methodologies, or byneedle-free technologies such as those described in Conry et al., 1999,Clin. Cancer Res., 5, 2330-2337 and PCT Publication No. WO 99/3 1262.

Pharmaceutically acceptable formulations of ALK-inhibiting therapeuticsinclude salts of the above described compounds, e.g., acid additionsalts, for example, salts of hydrochloric, hydrobromic, acetic acid, andbenzene sulfonic acid. A pharmacological composition or formulationrefers to a composition or formulation in a form suitable foradministration, e.g., systemic administration, into a cell or patient,including for example a human. Suitable forms, in part, depend upon theuse or the route of entry, for example oral, transdermal, or byinjection. Such forms should not prevent the composition or formulationfrom reaching a target cell. For example, pharmacological compositionsinjected into the blood stream should be soluble. Other factors areknown in the art, and include considerations such as toxicity and formsthat prevent the composition or formulation from exerting its effect.

Administration routes that lead to systemic absorption (e.g., systemicabsorption or accumulation of drugs in the blood stream followed bydistribution throughout the entire body), are desirable and include,without limitation: intravenous, subcutaneous, intraperitoneal,inhalation, oral, intrapulmonary and intramuscular. Each of theseadministration routes exposes the ALK-inhibiting therapeutic to anaccessible diseased tissue or tumor. The rate of entry of a drug intothe circulation has been shown to be a function of molecular weight orsize. The use of a liposome or other drug carrier comprising thecompounds of the instant invention can potentially localize the drug,for example, in certain tissue types, such as the tissues of thereticular endothelial system (RES). A liposome formulation that canfacilitate the association of drug with the surface of cells, such as,lymphocytes and macrophages is also useful. This approach can provideenhanced delivery of the drug to target cells by taking advantage of thespecificity of macrophage and lymphocyte immune recognition of abnormalcells, such as cancer cells.

By “pharmaceutically acceptable formulation” is meant, a composition orformulation that allows for the effective distribution of the nucleicacid molecules of the instant invention in the physical location mostsuitable for their desired activity. Nonlimiting examples of agentssuitable for formulation with the nucleic acid molecules of the instantinvention include: P-glycoprotein inhibitors (such as Pluronic P85),which can enhance entry of drugs into the CNS (Jolliet-Riant andTillement, 1999, Fundam. Clin. Phannacol., 13, 16-26); biodegradablepolymers, such as poly (DL-lactide-coglycolide) microspheres forsustained release delivery after intracerebral implantation (Emerich etal, 1999, Cell Transplant, 8, 47-58) (Alkermes, Inc. Cambridge, Mass.);and loaded nanoparticles, such as those made of polybutylcyanoacrylate,which can deliver drugs across the blood brain barrier and can alterneuronal uptake mechanisms (Prog Neuro-psychopharacol Biol Psychiatry,23, 941-949, 1999). Other non-limiting examples of delivery strategiesfor the ALK-inhibiting compounds useful in the method of the inventioninclude material described in Boado et al., 1998, J. Pharm. Sci., 87,1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge etal., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug DeliveryRev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26,4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

Therapeutic compositions comprising surface-modified liposomescontaining poly (ethylene glycol) lipids (PEG-modified, orlong-circulating liposomes or stealth liposomes) may also be suitablyemployed in the methods of the invention. These formulations offer amethod for increasing the accumulation of drugs in target tissues. Thisclass of drug carriers resists opsonization and elimination by themononuclear phagocytic system (MPS or RES), thereby enabling longerblood circulation times and enhanced tissue exposure for theencapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwataet al., Chem. Pharn. Bull. 1995, 43, 1005-1011). Such liposomes havebeen shown to accumulate selectively in tumors, presumably byextravasation and capture in the neovascularized target tissues (Lasicet al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim.Biophys. Acta, 1238, 86-90). The long-circulating liposomes enhance thepharmacokinetics and pharmacodynamics of DNA and RNA, particularlycompared to conventional cationic liposomes which are known toaccumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42,24864-24870; PCT Publication No. WO 96/10391; PCT Publication No. WO96/10390; and PCT Publication No. WO 96/10392). Long-circulatingliposomes are also likely to protect drugs from nuclease degradation toa greater extent compared to cationic liposomes, based on their abilityto avoid accumulation in metabolically aggressive MPS tissues such asthe liver and spleen.

Therapeutic compositions may include a pharmaceutically effective amountof the desired compounds in a pharmaceutically acceptable carrier ordiluent. Acceptable carriers or diluents for therapeutic use are wellknown in the pharmaceutical art, and are described, for example, inREMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Co. (A. R. Gennaroedit. 1985). For example, preservatives, stabilizers, dyes and flavoringagents can be provided. These include sodium benzoate, sorbic acid andesters of p-hydroxybenzoic acid. In addition, antioxidants andsuspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors that those skilled in the medical artswill recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kgbody weight/day of active ingredients is administered dependent uponpotency of the negatively charged polymer.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per patient perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient. It is understood that the specific dose level for anyparticular patient depends upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, and rate of excretion, drug combination and the severityof the particular disease undergoing therapy.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

An ALK-inhibiting therapeutic useful in the practice of the inventionmay comprise a single compound as described above, or a combination ofmultiple compounds, whether in the same class of inhibitor (e.g.,antibody inhibitor), or in different classes (e.g., antibody inhibitorsand small-molecule inhibitors). Such combination of compounds mayincrease the overall therapeutic effect in inhibiting the progression ofa fusion protein-expressing cancer. For example, Crizotinib (also knownas PF-02341066) produced by Pfizer, Inc. (see U.S. Pub. No.2008/0300273) may be administered alone, or in combination with otherCrizotinib analogues targeting ALK activity and/or small moleculeinhibitors of ALK, such as NVP-TAE684 produced by Novartis, Inc. Thetherapeutic composition may also comprise one or more non-specificchemotherapeutic agent in addition to one or more targeted inhibitors.Such combinations have recently been shown to provide a synergistictumor killing effect in many cancers. The effectiveness of suchcombinations in inhibiting ALK activity and tumor growth in vivo can beassessed according to standard methods.

The present invention is further directed to a method of monitoring theeffectiveness of a cancer therapy, such as kidney cancer therapy. Themethod involves detecting the expression levels or activity levels of apolypeptide with ALK kinase activity in the relevant biological sampleof the cancer patient prior to and after the cancer therapy. Theexpression levels of the polypeptide with ALK kinase activity arefurther quantified so as to be able to compare the values prior totherapy and after therapy. An increase or steady value in the expressionand/or levels of the polypeptide with ALK kinase activity after therapyas compared to before therapy indicates that the cancer therapy is noteffective. A decrease in the expression and/or activity level of thepolypeptide with ALK kinase activity (i.e., the level of the polypeptidewith ALK kinase activity is less after the therapy compared to prior totherapy) indicates an effective cancer therapy. In accordance with theinvention, the cancer therapy may include biological therapies, such assurgery, immunotherapeutics, chemotherapeutics, and radiation therapies,and targeted therapies, such as drugs or other substances intended toblock the growth and spread of cancer by interfering with specificmolecular in tumor growth. ALK inhibitor treatments, such as the use ofcrizotinib (PF02341066), may be monitored according to the presentinvention.

The invention further provides a method for determining whether acompound inhibits the progression of a cancer (e.g., a kidney cancer)driven by polypeptide with ALK kinase activity. In this embodiment, themethod comprises the step of determining whether the compound inhibitsthe expression and/or activity of the polypeptide with ALK kinaseactivity in the cancer. In some embodiments, inhibition of expressionand/or activity of the polypeptide with ALK kinase is determined byexamining a biological sample comprising cells from bone marrow, blood,or a tumor. In another embodiment, inhibition of expression and/oractivity of polypeptide with ALK kinase is determined using at least onedetection molecule as described herein.

The tested compound may be any type of therapeutic or composition asdescribed above. Methods for assessing the efficacy of a compound, bothin vitro and in vivo, are well established and known in the art. Forexample, a composition may be tested for ability to inhibit ALK in vitrousing a cell or cell extract in which ALK kinase is activated. A panelof compounds may be employed to test the specificity of the compound forALK (as opposed to other targets, such as EGFR or PDGFR).

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity to aprotein of interest, as described in PCT Publication No. WO 84/03564. Inthis method, as applied to ALK fusion polypeptides or full-length ALKpolypeptide, large numbers of different small test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. The test compounds are reacted with a polypeptide of theinvention, or fragments thereof, and washed. Bound polypeptide is thendetected by methods well known in the art. A purified polypeptide canalso be coated directly onto plates for use in the aforementioned drugscreening techniques. Alternatively, non-neutralizing antibodies can beused to capture the peptide and immobilize it on a solid support.

A compound found to be an effective inhibitor of ALK activity in vitromay then be examined for its ability to inhibit the progression of acancer expressing a polypeptide with kinase activity (such as kidneycancer), in vivo, using, for example, mammalian xenografts harboringhuman kidney tumors that are express a polypeptide with ALK kinaseactivity. In this procedure, cancer cell lines known to express apolypeptide with ALK kinase activity may be placed subcutaneously in ananimal (e.g., into a nude or SCID mouse, or other immune-compromisedanimal). The cells then grow into a tumor mass that may be visuallymonitored. The animal may then be treated with the drug. The effect ofthe drug treatment on tumor size may be externally observed. The animalis then sacrificed and the tumor removed for analysis by IHC and Westernblot. Similarly, mammalian bone marrow transplants may be prepared, bystandard methods, to examine drug response in hematological tumorsexpressing a mutant ALK kinase. In this way, the effects of the drug maybe observed in a biological setting most closely resembling a patient.The drug's ability to alter signaling in the tumor cells or surroundingstromal cells may be determined by analysis withphosphorylation-specific antibodies. The drug's effectiveness ininducing cell death or inhibition of cell proliferation may also beobserved by analysis with apoptosis specific markers such as cleavedcaspase 3 and cleaved PARP.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50. Insome embodiments, the compounds exhibit high therapeutic indices.

The following Examples are provided only to further illustrate theinvention, and are not intended to limit its scope, except as providedin the claims appended hereto. The present invention encompassesmodifications and variations of the methods taught herein which would beobvious to one of ordinary skill in the art. Materials, reagents and thelike to which reference is made are obtainable from commercial sources,unless otherwise noted.

Example 1 Identification of ALK or ALK Fusion Polypeptides in KidneyCancer Patients

Tissue microarrays comprised of samples of cancers of the breast,pancreas, prostate, bladder, endometrium, kidney and various metastaseswere obtained from BioChain Institute, Inc., Hayward, Calif.

Tissue arrays were deparaffinized through three changes of xylene for 5minutes each, then rehydrated through two changes of 100% ethanol and 2changes of 95% ethanol, each for 5 minutes. Slides were rinsed for 5minutes each in three changes of diH₂O, then were subjected to antigenretrieval in a Decloaking Chamber (Biocare Medical, Concord, Calif.) asfollows. Slides were immersed in 250 ml 1.0 mM EDTA, pH 8.0 in a 24slide holder from Tissue Tek. The Decloaking Chamber was filled with 500ml diH2O, the slide holder was placed in the chamber touching the heatshield, and retrieval was performed with the following settings as setby the manufacturer: SP1 125° C. for 30 seconds and SP2 90° C. for 10seconds. Slides were cooled on the bench for 10 minutes, rinsed indiH₂O, submerged in 3% H₂O₂ for 10 minutes, then washed twice in diH₂O.After blocking for 1 hour at room temperature in Tris bufferedsaline+0.5% Tween-20 (TBST)/5% goat serum in a humidified chamber,slides were incubated overnight at 4° C. with ALK (D5F3) XP® Rabbit mAb(Cell Signaling Technology, Inc., Danvers, Mass.; Catalog No. 3633) at9.8 pg/ml diluted in SignalStain® Antibody Diluent (Catalog No. 8112Cell Signaling Technology, Inc.). After washing three times in TBST,detection was performed with Envision+(DAKO, Carpinteria, Calif.) with a30 minute incubation at room temperature in a humidified chamber. Afterwashing three times in TBST slides were exposed to NovaRed (VectorLaboratories, Burlingame, Calif.) prepared per the manufacturer'sinstructions. Slides were developed for 1 minute then rinsed in diH₂O.Slides were counterstained by incubating in hematoxylin (Ready to UseInvitrogen, Carlsbad, Calif.; Catalog #00-8011) for 1 minute, rinsed for30 seconds in diH2O, incubated for 20 seconds in bluing reagent (RichardAllan Scientific, Catalog #7301), then finally washed for 30 seconds indiH2O. Slides were dehydrated in 2 changes of 95% ethanol for 20 secondseach and 2 changes of 100% ethanol for 2 minutes each. Slides werecleared in 2 changes of xylene for 20 seconds each, then air dried.Coverslips were mounted using VectaMount (Vector Laboratories,Burlingame, Calif.). Slides were air dried, then evaluated under themicroscope.

Staining was observed in 1 lymphoma in the kidney and 1 squamous cellcarcinoma of the kidney.

Additional arrays of kidney carcinomas and normal kidney tissue (331carcinomas and 26 normal tissues) were acquired from BioChain Institute,Inc., Hayward, Calif. and Folio Biosciences Columbus, Ohio and stainedwith ALK (D5F3) XP® Rabbit mAb as described above. Staining was observedin 1 squamous cell carcinoma of the kidney (FIG. 1) and 1 granular cellcarcinoma (FIG. 2).

Total kidney cancer cases stained, including lymphomas: 331Positive cases identified: 4

Frequency: 1.2%

Total cancers stained, excluding lymphoma: 327Positive cases identified: 3

Frequency: 0.9%

Breakdown of frequency by cancer type:

TABLE 1 Cancer Cases Positive Staining Frequency Granular cell 26 1 3.8% Squamous cell 9 2 22.2% Lymphoma 4 1  25%

Example 2 Evaluation of ALK in Kidney Using FISH Analysis

ALK was analyzed by fluorescent in situ hybridization (FISH) in alymphoma tissue of the kidney (Z7020052 I6/J6) and a squamous cellcarcinoma tissue of the kidney (Z6020052 G8/H8) with the use of abreak-apart probe specific to the ALK locus (Vysis LSI ALK Dual Color,Break Apart Rearrangement Probe; Abbott Molecular). The Vysis probecontains two differently labeled probes on opposite sides of thebreakpoint of the ALK gene: one approximately 250 kb probe for thetelomeric side of the ALK breakpoint is labeled with SpectrumOrange andthe centromeric probe is approximately 300 kb and labeled withSpectrumGreen. When hybridized with the LSI ALK Dual Color, Break ApartRearrangement Probe, the 2p23 ALK region in its native state is seen astwo immediately adjacent or fused orange/green (yellow) signals.Paraffin embedded tissue sections were re-hydrated and incubated for 1hour in TE buffer pH 8 in boiling water. Sections were digested withpepsin Digest All III (Invitrogen) at 37° C. for 20-40 minutes. Slideswere then fixed with NBF for 1 minute and then dehydrated. The probe andtissue were then co-denatured at 93° C. for 3 minutes and then allowedto incubate at 37° C. for 18 hours. After washing,4′,6-diamidino-2-phenylindole (DAPI; mg/ml) in Vectashield mountingmedium (Vector Laboratories, Burlingame, Calif.) was applied for nuclearcounterstaining. Cytogenetic rearrangement of the ALK locus wereconfirmed by FISH in the kidney lymphoma and squamous cell carcinoma ofthe kidney (FIG. 3). The results (with IHC results of the same tissues)are summarized in Table 2.

TABLE 2 TUMOR ID TYPE ALK IHC ALK FISH Z7020052 I6/J6 KidneyLymphoma + + Z6020052 G8/H8 Kidney Squamous + +

Example 3 Evaluation of MDCK for ALK and c-Met in Western BlotExperiment

U.S. Patent Application Publication No. 2008/0300273, hereinincorporated by reference, describes the application of the c-Met andALK inhibitor, crizotinib (PF-02341066) to Madin-Darby Canine Kidney(MDCK) cell lines to reduce HGF-stimulated cell scattering.

HGF-stimulated MDCK cell lines were evaluated to assess whethercrizotinib was acting by inhibition of c-Met or ALK using antibodiesspecific for either c-Met, ALK or ALK fusion polypeptides.

MDCK were maintained in Dulbecco's modified Eagle's medium supplementedwith 10% fetal bovine serum. The cells were serum-starved overnight andthen either stimulated with hepatocyte growth factor (HGF) (50 ng/ml) at37° C. for 5 min or 24 h, or serum-starved for an additional 5 min or 24h. Cells were then washed in ice-cold PBS, treated with 1× Cell LysisBuffer (20 mM Tris-HCl (pH7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1%Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mMNa_(A)VO₄, 1 ug/mL leupeptin, 1% SDS and 40 mM DTT) for 5 min on ice,and then scraped. H3122 cells (expressing EML4-ALK fusion polypeptide)were used as a positive control for ALK. MKN45 cells were used as apositive control for pMet.

The MDCK, H3122, and MKN45 cell lysates were analyzed by Western Blot.The membranes were probed with the following antibodies: pMet Y1234/5antibody (Cell Signaling Technology, #3077), Met (25H2) mouse monoclonalantibody (Cell Signaling Technology, #3127), pALK Y1278/82/83 antibody(Cell Signaling Technology #3983). ALK (D5F3) XP®RmAb (Cell SignalingTechnology #3633), and Beta Actin (Cell Signaling Technology #4970).

The results of the Western blot are represented by a 1 second exposure(FIG. 4A) and a 15 second exposure (FIG. 4B). As shown, the MDCK cells(lanes 1, 2, 3, and 4) are positive for Met (Cell Signaling Technology#3127), but not ALK (Cell Signaling Technology #3633).

These results demonstrate that MDCK cells stimulated by HGF do notexpress ALK. Therefore, to the extent crizotinib inhibits MDCK cellscattering in response to HGF stimulation, the mechanism of actionappears to be that it inhibits c-Met/HGFR activity.

Therefore, inhibition of MDCK cell lines in scattering assays, asdescribed in the US Patent Publication 2008/0300273, appears to beacting through Met-related activity and not ALK activity.

Example 4 Detection of ALK or ALK Fusion Polypeptide Expression in aHuman Cancer Sample Using PCR Assay

The presence of ALK and/or an ALK fusion polypeptide of the invention incancer sample is detected using RT-PCR. These methods have beenpreviously described. See, e.g., Cools et al., N. Engl. J. Med. 348:1201-1214 (2003).

PCR Assay

To confirm the existence of an EML4-ALK fusion gene in genomic DNA,standard PCR methods known in the art for detecting the relevanttranslocation may be used.

Genomic PCR is done with 50 to 100 ng of DNA in a 25 μL reactioncontaining LongAmp Taq DNA polymerase (New England Biolabs, Ipswich,Mass.) under the following conditions: 3 min at 95° C. followed by 30cycles of 10 s at 95° C., 1 min at 55° C., and 10 min at 68° C. plus afinal extension for 20 min at 68° C. The genomic fusion points for theE13; A20 variant are identified using forward PCR primer Fusion-genome-Sor a primer residing in EML4 intron 13 (AGGA GAGAAAGAGCTGCAGTG) (SEQ IDNO: 7) and reverse primer Fusion-genome-AS or a primer located in ALKintron 19 (GCTCTGAACCTTTCCATCATACTT) (SEQ ID NO:8). For the detection ofthe E20; A20 and E21; A20 variants, the forward primers are placedwithin EML4 exon 20 (ACTGGTCCCCAGACAACAAG) (SEQ ID NO:9) or intron 20(TTACTCTGTCAAATTGATGCTGCT) (SEQ ID NO:10), whereas the reverse primer isFusion-genome-AS. The PCR products are resolved on agarose gel; if theyappear specific, the original PCR product is used for direct sequencing.However, if additional nonspecific fragments are present, the desiredfragments are excised, gel purified, cloned, and sequenced.

A fusion partner of ALK is determined by performing RNA ligase-mediatedrapid amplification of 5′ and 3′ cDNA ends with GeneRacer kit(Invitrogen). First-strand cDNA is amplified with Advantage HD DNApolymerase mix (Clontech) using GeneRacer 5′ primer and ALK-6R primer(CATGAGGAAATCCAGTTCGTCCTG) (SEQ ID NO:11). Subsequent nested PCR is doneusing GeneRacer 5′ nested primer and ALK-2R primer(GAGGTCTTGCCAGCAAAGCAGTAG) (SEQ ID NO:12). Amplification products aregel purified with QIAquick gel extraction (Qiagen) and cloned usingpCR4-TOPO TA Cloning (Invitrogen). Sequencing is done using 3730xl DNAAnalyzer (Applied Biosystems). Sequencing products may be analyzed withSequencer software (Gene Codes). Basic Local Alignment and Search Toolagainst the BLAT database4 is used to determine the identity of unknownsequences

To determine whether EML4-ALK gene product is expressed, RT-PCR isperformed on RNA extracted from the kidney cancer cell samples ofpatients. RT-PCR is carried out using One Step RT-PCR (Qiagen) andprimers previously described in (Soda et al., Nature 2007; 448:561-6.Choi Y L, Takeuchi K, Soda M, et al. Identification of novel isoforms ofthe EML4-ALK transforming gene in non-small cell lung cancer. Cancer Res2008; 68:4971-76. PCR conditions for the detection of EML4-ALK fusiontranscript include cDNA synthesis at 50° C. for 30 min, denaturation at95° C. for 15 min, 40 cycles consisting of denaturation at 95° C. for 30s, annealing at 60° C. for 30 s, and strand elongation at 72° C. for 1min and a final elongation step at 72° C. for 10 min. As an internalcontrol, primers for the glyceraldehyde-3-phosphate dehydrogenase(GAPDH; AACGACCACTTTGTCAAGCTC (SEQ ID NO:13) andCTCTCTTCCTCTTGTGCTCTTGC) (SEQ ID NO:14) are used. Twenty cycles ofamplification are performed. PCR products are resolved on agarose geland their sizes are determined by using Trackit 1 kb Plus DNA ladder(Invitrogen). Fragments representing EML4-ALK fusion product areexcised, gel purified, cloned, and sequenced.

Expression of ALK Protein or ALK Fusion Polypeptide

The expression of ALK and ALK fusion polypeptides is investigated byWestern blotting and immunoprecipitation with anti-ALK antibodies.

Cell lysates are prepared from kidney cancer cells by removing media andrinsing the cells with ice-cold PBS. The PBS is removed and 0.5 mlice-cold 1× cell lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mMEDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Sodium pyrophosphate, 1 mMβ-glycerophosphate, 1 mM Na₃VO₄, 1 pg/ml Leupeptin) is added to eachplate and incubated. The cells are transferred to microcentrifuge tubesand samples are sonicated on ice three times for 5 second each andmicrocentrifuged for 10 min at 14,000×g.

The cell lysate is incubated with the primary antibodies specific forALK protein, pALK Y1278/82/83 antibody (CST #3893) and ALK (D5F3)XP™RmAb (CST #3633) overnight at 4° C. Protein A or G agarose beads areadded and incubated with gentle rocking for 1-3 hours at 4° C. Thesample is microcentrifuged for 30 s at 4° C., washed, and resuspendedwith 20 μl 3×SDS sample buffer. The sample is loaded on an SDS-PAGE geland analyzed by Western blotting.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

1.-30. (canceled)
 31. A method for treating a human patient with kidneycancer or suspected kidney cancer driven by an ALK fusion polypeptidewith ALK kinase activity, said method comprising the step ofadministering a composition comprising a therapeutically effectiveamount of an ALK-inhibiting therapeutic to the patient. 32.-34.(canceled)
 35. A method for treating a human patient having kidneycancer or suspected kidney cancer comprising the steps of: (a) detectingthe presence or activity of an ALK fusion polypeptide with ALK kinaseactivity in a biological sample of the kidney cancer or suspected kidneycancer of the patient; and (b) administering a therapeutically effectiveamount of a composition comprising an ALK-inhibiting therapeutic to themammalian patient.
 36. (canceled)
 37. The method of claim 31, whereinthe ALK-inhibiting therapeutic is selected from the group consisting ofPF-02341066, NVT TAE-684, and AP26113.
 38. The method of claim 31,wherein the ALK-inhibiting therapeutic is selected from the groupconsisting of CEP-14083, CEP-14513, CEP11988, WHI-P131 and WHI-P154. 39.The method of claim 35, wherein the ALK-inhibiting therapeutic isselected from the group consisting of PF-02341066, NVT TAE-684, andAP26113.
 40. The method of claim 35, wherein the ALK-inhibitingtherapeutic is selected from the group consisting of CEP-14083,CEP-14513, CEP11988, WHI-P131 and WHI-P154.
 41. A method for treating ahuman subject having kidney cancer that expresses a human AnaplasticLymphoma Kinase (ALK) fusion polypeptide, comprising administering tothe human subject a small molecule that a) binds to the ATP-bindingcleft and b) directly inhibits the kinase activity of the ALK fusionpolypeptide, in an amount effective to treat the kidney cancer, whereinthe −ALK fusion polypeptide comprises the ALK kinase domain as set forthin SEQ ID NO:
 3. 42. The method of claim 39, wherein the ALK fusionpolypeptide also comprises the intracytoplasmic domain of ALK as setforth in SEQ ID NO:
 4. 43. The method of claim 41, wherein the ALKfusion polypeptide is an Echinoderm Microtubule-Associated Protein-Like4 (EML4)-ALK fusion polypeptide.
 44. The method of claim 41, whereinsaid kidney cancer is a granular cell cancer or a squamous cell cancer.45. The method of claim 43, wherein said kidney cancer is a granularcell cancer or a squamous cell cancer.